Method for treating fibrosis using sirna and a retinoid-lipid drug carrier

ABSTRACT

What is described is a method for treating a fibrotic disease by administering a pharmaceutical composition comprising a drug carrier, which comprises a lipid and a retinoid, and a double-stranded nucleic acid molecule, which comprises an antisense sequence to mRNA encoding human hsp47.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/294,021, filed Jun. 2, 2014, which is a continuation of U.S. patentapplication Ser. No. 13/492,594, filed Jun. 8, 2012, issued as U.S. Pat.No. 8,741,867, which claims benefit under 35 U.S.C. §119(e) of U.S.Provisional patent application No. 61/494,832, filed Jun. 8, 2011, theentire contents of which are incorporated herein by reference in theirentirety.

TECHNICAL FIELD

Provided herein are pharmaceutical compositions comprisingretinoid-liposomes for enhancing the modulation of hsp47 expression bysiRNA.

BACKGROUND

Fibrosis of the liver can be caused by activated hepatic stellate cells(HSC), resulting in a plurality of types of collagen molecules andfibronectin being deposited on interstitial tissue. This can lead tohepatic cirrhosis, hepatic failure, and/or hepatocellular carcinoma.Further, chronic pancreatitis develops as a result of pancreaticfibrosis by the same mechanism as that for hepatic fibrosis (Madro, etal., 2004; Med Sci Monit. 10:RA166-70; Jaster, 2004, Mol Cancer. 6:26).Furthermore, stellate cells are present in disorders of the vocal cordand larynx such as vocal cord scarring, vocal cord mucosal fibrosis, andlaryngeal fibrosis. To prevent or treat fibrosis in these organs andelsewhere in the body, there is a desire for the development of a drugcarrier and drug carrier kit.

Stellate cells are one of the important target candidates for treatingfibrosis (Fallowfield et al., 2004, Expert Opin Ther Targets. 8:423-35;Pinzani, et al., 2004, Dig Liver Dis. 36:231-42). During fibrosis,stellate cells are activated by cytokines from nearby cells to producemany factors that cause hepatic fibrosis. Stellate cells store vitaminA, and belong to the myofibroblast family.

Therapeutic methods to prevent or treat fibrosis attempt to controlcollagen metabolism, promotion of the collagen degradation system, andinhibition of activation of stellate cells. However, in all these cases,the low specificity of action and/or the low organ specificity, limitedefficacy and adverse side effects create problems.

Inhibition of collagen protein synthesis has not been established as atherapeutic method. The potency of molecules targeting collagenproduction is limited due to the possibility of causing side effects.Inhibiting collagen production directly provides another therapeuticmethod to prevent or treat fibrosis. Such a method requires controllingone or more of the various types of collagen Types I to IV. A method foraccomplishing this may be through heat shock protein47 (HSP47), acollagen-specific molecular chaperone that is essential forintracellular transport and molecular maturation necessary for varioustypes of collagen. Therefore, if the function of HSP47 can bespecifically controlled in stellate cells, there is a possibility ofinhibiting hepatic fibrosis.

SUMMARY

The present description relates to a drug carrier and a drug carrier kitthat enable a diagnostic and/or therapeutic drug to be specificallytransported to stellate cells. The drug carrier in the presentdescription may be selected from polymer micelles, liposomes, emulsions,microspheres, and nanosphere forms, and by bonding thereto or includingtherein a retinoid or retinoid conjugate and a therapeutic drug that canbe transported specifically to HSC. Retinoids include vitamin A,retinal, retinoic acid, saturated Vitamin A, tretinoin, adapalene, orretinol palmitate, and fenretinide (4-HPR). Furthermore, by preparingthe drug carrier to include one molecule or a plurality of moleculesselected from TGFβ activity inhibitors such as a truncated TGFβ type IIreceptor and a soluble TGFβ type II receptor, growth factor preparationssuch as HGF, MMP production promoters such as an MMP gene-containingadenovirus vector, a cell activation inhibitors and/or growth inhibitorsincluding a PPARγ-ligand, an angiotensin-II type I receptor antagonist,a PDGF tyrosine kinase inhibitor, and a sodium channel inhibitor such asamiloride, and apoptosis inducers such as compound 861 and gliotoxin;and by administering it for example, orally, parenterally, intravenouslyor intraperitoneally to a patient having a risk of fibrosis or fibrosissymptoms, or patients having various fibrosis-related disorders such as,for example, hepatic cirrhosis, hepatic failure, liver cancer, orchronic pancreatitis, the activation of stellate cells can besuppressed, and thereby preventing, inhibiting or improving the fibrosisand/or fibrosis-related disease conditions in said patient.Alternatively, or in addition thereto, by using the drug carrier whichencloses therein a ribozyme, an antisense RNA, or an siRNA thatspecifically inhibits HSP47 or TIMP, which is an MMP inhibitor,secretion of type I to IV collagens can be simultaneously inhibited, andas a result fibrogenesis can be inhibited effectively.

An embodiment of the description is a pharmaceutical compositioncomprising a double-stranded nucleic acid molecule comprising a sensestrand and an antisense strand wherein the sense and antisense strandsare selected from the oligonucleotides described as SERPINH1_2 (SEQ IDNOS: 60 and 127), SERPINH1_45a (SEQ ID NOS: 98 and 165), and SERPINH1_51(SEQ ID NOS: 101 and 168) in Table 4, infra, and a drug carriercomprising a mixture of a lipid vesicle and a retinoid or a retinoidconjugate. The retinoid can be one or more of the following: vitamin A,retinoic acid, saturated Vitamin A, retinal, tretinoin, adapalene,retinol palmitate, or fenretinide. Preferably, the retinoid comprises aconjugate of retinoic acid, most preferably a retinoid-PEG conjugate.The lipid vesicle can be comprised of a bilayer of lipid molecules, andcan further be comprised of the retinoid. The retinoid is preferably ata concentration of 0.2 to 20 wt % in the drug carrier. The lipid vesiclecan be comprised of an interior surface that encapsulates the interiorof the lipid vesicle, and an exterior surface that is accessible to anaqueous medium outside of the lipid vesicle. The retinoid may beassociated with the lipid bilayer. The double-stranded nucleic acid canbe exposed on the exterior surface of the lipid vesicle.

In some embodiments, the double-stranded nucleic acid molecule includesan antisense strand having SEQ ID NO:127 and comprising 2′-O-methylsugar (2′OMe)-modified ribonucleotides; a 2′-5′-ribonucleotide in atleast one of positions 1, 5, 6, or 7; and a 3′-terminal non-nucleotidemoiety covalently attached to the 3′-terminus; and a sense strand havingSEQ ID NO:60 and comprising at least one 2′-5′-ribonucleotide or 2′OMemodified ribonucleotide; a non-nucleotide moiety covalently attached atthe 3′-terminus; and an inverted abasic moiety covalently attached atthe 5′-terminus. In preferred embodiments the antisense strand is SEQ IDNO:127 and comprises 2′OMe modified ribonucleotides at positions 3, 5,9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and anon-nucleotide moiety covalently attached at the 3′-terminus; and thesense strand is SEQ ID NO:60 and comprises five consecutive2′-5′-ribonucleotides in the 3′-terminal positions 15, 16, 17, 18, and19; a non-nucleotide moiety covalently attached at the 3′-terminus; andan inverted abasic moiety covalently attached at the 5′-terminus. Insome embodiments the double-stranded nucleic acid molecule furtherincludes a 2′OMe modified ribonucleotide or a 2′-5′-ribonucleotide atposition 1 of the antisense strand. (All reference herein to nucleotidepositions are expressed based on the 5′>3′ direction of theoligonucleotide for both sense and antisense strands of thedouble-stranded nucleic acid molecule.)

In various embodiments, the sense strand is SEQ ID NO:98 and comprises2′-5′-ribonucleotides in positions at the 3′-terminus; a non-nucleotidemoiety covalently attached at the 3′-terminus; and an inverted abasicmoiety covalently attached at the 5′-terminus; and the antisense strandis SEQ ID NO:165 and comprises 2′OMe modified ribonucleotides; a2′-5′-ribonucleotide in at least one of positions 5, 6 or 7; and anon-nucleotide moiety covalently attached at the 3′-terminus. Inpreferred embodiments the sense strand is SEQ ID NO:98 and comprises2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; a C3-OH 3′moiety covalently attached at the 3′-terminus; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand is SEQ ID NO:165 and comprises 2′OMe modifiedribonucleotides in positions 4, 6, 8, 11, 13, 15, 17, and 19; a2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH moiety covalentlyattached at the 3′-terminus. In some embodiments the double-strandednucleic acid further comprises a 2′OMe modified ribonucleotide inposition 2.

In various embodiments, the sense strand is SEQ ID NO:101 and comprises2′OMe modified ribonucleotides; an optional 2′-5′-ribonucleotide in oneof position 9 or 10; a non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic moiety covalently attached at the5′-terminus; and the antisense strand is SEQ ID NO:168 and comprises2′OMe modified ribonucleotides; a 2′-5′-ribonucleotide in at least oneof positions 5, 6, or 7; and a non-nucleotide moiety covalently attachedat the 3′-terminus. In preferred embodiments the sense strand is SEQ IDNO:101 and comprises 2′OMe modified ribonucleotides in positions 4, 11,13, and 17; a 2′-5′-ribonucleotide in position 9; a C3OH non-nucleotidemoiety covalently attached at the 3′-terminus; and inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand is SEQ ID NO:168 and comprises 2′OMe modifiedribonucleotides in positions 1, 4, 8, 11 and 15; a 2′-5′-ribonucleotidein position 6; and a C3Pi-C3OH moiety covalently attached at the3′-terminus. In certain embodiments the double-stranded nucleic acidmolecule further comprises a 2′OMe modified ribonucleotide in position13 in the antisense strand and or in position 2 in the sense strand.

Another aspect is a pharmaceutical composition comprising adouble-stranded nucleic acid molecule and drug carrier comprising amixture of a retinoid and a lipid, wherein the double-strandedoligonucleotide compound comprising the structure (A1):

(A1) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y)-z″ 5′ (sensestrand)wherein each of N and N′ is a nucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of Z and Z′ is independently present or absent, but ifpresent independently includes 1-5 consecutive nucleotides ornon-nucleotide moieties or a combination thereof covalently attached atthe 3′-terminus of the strand in which it is present;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of (N′)_(y);wherein each of x and y is independently an integer between 18 and 40;wherein the sequence of (N′)_(y) has complementary to the sequence of(N)_(x); and wherein (N)_(x) includes an antisense sequence to the mRNAcoding sequence for human hsp47 exemplified by SEQ ID NO:1, which isshown as follows.

ucuuuggcuu uuuuuggcgg agcuggggcg cccuccggaa gcguuuccaa cuuuccagaa 60guuucucggg acgggcagga gggggugggg acugccauau auagaucccg ggagcagggg 120agcgggcuaa gaguagaauc gugucgcggc ucgagagcga gagucacguc ccggcgcuag 180cccagcccga cccaggccca ccguggugca cgcaaaccac uuccuggcca ugcgcucccu 240ccugcuucuc agcgccuucu gccuccugga ggcggcccug gccgccgagg ugaagaaacc 300ugcagccgca gcagcuccug gcacugcgga gaaguugagc cccaaggcgg ccacgcuugc 360cgagcgcagc gccggccugg ccuucagcuu guaccaggcc auggccaagg accaggcagu 420ggagaacauc cuggugucac ccgugguggu ggccucgucg cuagggcucg ugucgcuggg 480cggcaaggcg accacggcgu cgcaggccaa ggcagugcug agcgccgagc agcugcgcga 540cgaggaggug cacgccggcc ugggcgagcu gcugcgcuca cucagcaacu ccacggcgcg 600caacgugacc uggaagcugg gcagccgacu guacggaccc agcucaguga gcuucgcuga 660ugacuucgug cgcagcagca agcagcacua caacugcgag cacuccaaga ucaacuuccg 720cgacaagcgc agcgcgcugc aguccaucaa cgagugggcc gcgcagacca ccgacggcaa 780gcugcccgag gucaccaagg acguggagcg cacggacggc gcccugcuag ucaacgccau 840guucuucaag ccacacuggg augagaaauu ccaccacaag augguggaca accguggcuu 900cauggugacu cgguccuaua ccgugggugu caugaugaug caccggacag gccucuacaa 960cuacuacgac gacgagaagg aaaagcugca aaucguggag augccccugg cccacaagcu 1020cuccagccuc aucauccuca ugccccauca cguggagccu cucgagcgcc uugaaaagcu 1080gcuaaccaaa gagcagcuga agaucuggau ggggaagaug cagaagaagg cuguugccau 1140cuccuugccc aagggugugg uggaggugac ccaugaccug cagaaacacc uggcugggcu 1200gggccugacu gaggccauug acaagaacaa ggccgacuug ucacgcaugu caggcaagaa 1260ggaccuguac cuggccagcg uguuccacgc caccgccuuu gaguuggaca cagauggcaa 1320ccccuuugac caggacaucu acgggcgcga ggagcugcgc agccccaagc uguucuacgc 1380cgaccacccc uucaucuucc uagugcggga cacccaaagc ggcucccugc uauucauugg 1440gcgccugguc cggccuaagg gugacaagau gcgagacgag uuauagggcc ucagggugca 1500cacaggaugg caggaggcau ccaaaggcuc cugagacaca ugggugcuau ugggguuggg 1560ggggagguga gguaccagcc uuggauacuc cauggggugg ggguggaaaa acagaccggg 1620guucccgugu gccugagcgg accuucccag cuagaauuca cuccacuugg acaugggccc 1680cagauaccau gaugcugagc ccggaaacuc cacauccugu gggaccuggg ccauagucau 1740ucugccugcc cugaaagucc cagaucaagc cugccucaau caguauucau auuuauagcc 1800agguaccuuc ucaccuguga gaccaaauug agcuaggggg gucagccagc ccucuucuga 1860cacuaaaaca ccucagcugc cuccccagcu cuaucccaac cucucccaac uauaaaacua 1920ggugcugcag ccccugggac caggcacccc cagaaugacc uggccgcagu gaggcggauu 1980gagaaggagc ucccaggagg ggcuucuggg cagacucugg ucaagaagca ucgugucugg 2040cguugugggg augaacuuuu uguuuuguuu cuuccuuuuu uaguucuuca aagauaggga 2100gggaaggggg aacaugagcc uuuguugcua ucaauccaag aacuuauuug uacauuuuuu 2160uuuucaauaa aacuuuucca augacauuuu guuggagcgu ggaaaaaa 2208

Compositions, methods and kits for modulating expression of target genesare provided herein. In various aspects and embodiments, compositions,methods and kits provided herein modulate expression of heat shockprotein 47 (hsp47), also known as SERPINH1 (SEQ ID NO:1). Thecompositions, methods and kits may involve use of nucleic acid molecules(for example, short interfering nucleic acid (siNA), short interferingRNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or shorthairpin RNA (shRNA)) that bind a nucleotide sequence (such as an mRNAsequence) encoding hsp47, SEQ ID NO:1. In certain preferred embodiments,the compositions, methods and kits disclosed herein inhibit expressionof hsp47. For example, siNA molecules (e.g., RNA-induced silencingcomplex (RISC) length dsNA molecules or Dicer length dsNA molecules) areprovided that reduce or inhibit hsp47 expression. Also provided arecompositions, methods and kits for treating and/or preventing diseases,conditions or disorders associated with hsp47, such as liver fibrosis,cirrhosis, pulmonary fibrosis including lung fibrosis (includinginterstitial lung fibrosis (ILF)), kidney fibrosis resulting from anycondition (e.g., chronic kidney disease (CKD) including End-Stage RenalDisease (ESRD)), peritoneal fibrosis, chronic hepatic damage,fibrillogenesis, fibrotic diseases in other organs, abnormal scarring(keloids) associated with all possible types of skin injury accidentaland jatrogenic (operations); scleroderma; cardiofibrosis, failure ofglaucoma filtering operation; and intestinal adhesions.

In one aspect, provided are the pharmaceutical compositions, above,comprising nucleic acid molecules (e.g., siNA molecules) as a componentof a pharmaceutical formulation in which the nucleic acid moleculeincludes a sense strand and an antisense strand; each strand of thenucleic acid molecule is independently 15 to 49 nucleotides in length; a15 to 49 nucleotide sequence of the antisense strand is complementary toa sequence of an mRNA encoding human hsp47 (e.g., SEQ ID NO: 1); and a15 to 49 nucleotide sequence of the sense strand is complementary to thea sequence of the antisense strand and includes a 15 to 49 nucleotidesequence of an mRNA encoding human hsp47 (e.g., SEQ ID NO: 1).

In certain embodiments, the sequence of the antisense strand that iscomplementary to a sequence of an mRNA encoding human hsp47 includes asequence complimentary to a sequence between nucleotides 600-800; or801-899; or 900-1000; or 1001-1300 of SEQ ID NO: 1; or betweennucleotides 650-730; or 900-975 of SEQ ID NO: 1. In some embodiments,the antisense strand includes a sequence that is complementary to asequence of an mRNA encoding human hsp47 corresponding to nucleotides674-693 of SEQ ID NO: 1 or a portion thereof; or nucleotides 698-716 ofSEQ ID NO: 1 or a portion thereof; or nucleotides 698-722 of SEQ ID NO:1 or a portion thereof; or nucleotides 701-720 of SEQ ID NO: 1 or aportion thereof; or nucleotides 920-939 of SEQ ID NO: 1 or a portionthereof; or nucleotides 963-982 of SEQ ID NO: 1 or a portion thereof; ornucleotides 947-972 of SEQ ID NO: 1 or a portion thereof; or nucleotides948-966 of SEQ ID NO: 1 or a portion thereof; or nucleotides 945-969 ofSEQ ID NO: 1 or a portion thereof; or nucleotides 945-963 of SEQ ID NO:1 or a portion thereof.

In certain embodiments, the antisense strand of a nucleic acid molecule(e.g., a siNA molecule) as disclosed herein as a component of apharmaceutical formulation includes a sequence corresponding to SEQ IDNO: 4 or a portion thereof; or SEQ ID NO: 6 or a portion thereof; or SEQID NO: 8 or a portion thereof; or SEQ ID NO: 10 or a portion thereof; orSEQ ID NO: 12 or a portion thereof; or SEQ ID NO: 14 or a portionthereof; or SEQ ID NO: 16 or a portion thereof; or SEQ ID NO: 18 or aportion thereof; or SEQ ID NO: 20 or a portion thereof; or SEQ ID NO: 22or a portion thereof; or SEQ ID NO: 24 or a portion thereof; or SEQ IDNO: 26 or a portion thereof; or SEQ ID NO: 28 or a portion thereof; orSEQ ID NO: 30 or a portion thereof; or SEQ ID NO: 32 or a portionthereof; or SEQ ID NO: 34 or a portion thereof; or SEQ ID NO: 36 or aportion thereof; or SEQ ID NO: 38 or a portion thereof; or SEQ ID NO: 40or a portion thereof; or SEQ ID NO: 42 or a portion thereof; or SEQ IDNO: 44 or a portion thereof; or SEQ ID NO: 46 or a portion thereof; orSEQ ID NO: 48 or a portion thereof; or SEQ ID NO: 50 or a portionthereof; or SEQ ID NO: 52 or a portion thereof; or SEQ ID NO: 54 or aportion thereof; or SEQ ID NO: 56 or a portion thereof; or SEQ ID NO: 58or a portion thereof. In certain embodiments, the sense strand of anucleic acid molecule (e.g., a siNA molecule) as disclosed hereinincludes a sequence corresponding to SEQ ID NO: 3 or a portion thereof;or SEQ ID NO: 5 or a portion thereof; or SEQ ID NO: 7 or a portionthereof; or SEQ ID NO: 9 or a portion thereof; or SEQ ID NO: 11 or aportion thereof; or SEQ ID NO: 13 or a portion thereof; or SEQ ID NO: 15or a portion thereof; or SEQ ID NO: 17 or a portion thereof; or SEQ IDNO: 19 or a portion thereof; or SEQ ID NO: 21 or a portion thereof; orSEQ ID NO: 23 or a portion thereof; or SEQ ID NO: 25 or a portionthereof; or SEQ ID NO: 27 or a portion thereof; or SEQ ID NO: 29 or aportion thereof; or SEQ ID NO: 31 or a portion thereof; or SEQ ID NO: 33or a portion thereof; or SEQ ID NO: 35 or a portion thereof; or SEQ IDNO: 37 or a portion thereof; or SEQ ID NO: 39 or a portion thereof; orSEQ ID NO: 41 or a portion thereof; or SEQ ID NO: 43 or a portionthereof; or SEQ ID NO: 45 or a portion thereof; or SEQ ID NO: 47 or aportion thereof; or SEQ ID NO: 49 or a portion thereof; or SEQ ID NO: 51or a portion thereof; or SEQ ID NO: 53 or a portion thereof; or SEQ IDNO: 55 or a portion thereof; or SEQ ID NO: 57 or a portion thereof.

In certain preferred embodiments, the antisense strand of a nucleic acidmolecule (e.g., a siNA molecule) as disclosed herein as a component of apharmaceutical formulation includes a sequence corresponding to any oneof the antisense sequences shown on Table 4. In certain preferredembodiments the antisense strand and the sense strand are selected fromthe sequence pairs shown in Table 4. In some embodiments the antisenseand sense strands are selected from the sequence pairs set forth inSERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58 andSERPINH1_88. In some embodiments the antisense and sense strands areselected from the sequence pairs set forth in SERPINH1_4 (SEQ ID NOS:195and 220), SERPINH1_12 (SEQ ID NOS:196 and 221), SERPINH1_30 (SEQ IDNOS:199 and 224), and SERPINH1_58 (SEQ ID NOS:208 and 233).

In some embodiments, the antisense and sense strands of a nucleic acidmolecule (e.g., a siNA molecule) as disclosed herein as a component of apharmaceutical formulation includes the sequence pairs set forth inSERPINH1_4 (SEQ ID NOS:195 and 220). In some embodiments of a nucleicacid molecule (e.g., a siNA molecule) as disclosed herein includes theantisense and sense strands of the sequence pairs set forth inSERPINH1_12 (SEQ ID NOS:196 and 221). In some embodiments the antisenseand sense strands of a nucleic acid molecule (e.g., a siNA molecule) asdisclosed herein includes the sequence pairs set forth in SERPINH1_30(SEQ ID NOS:199 and 224). In some embodiments of a nucleic acid molecule(e.g., a siNA molecule) as disclosed herein includes the antisense andsense strands of the sequence pairs set forth in SERPINH1_58 (SEQ IDNOS:208 and 233).

In certain embodiments, the antisense strand of a nucleic acid molecule(e.g., a siNA molecule) as disclosed herein as a component of apharmaceutical formulation includes a sequence corresponding to any oneof the antisense sequences shown on any one of Tables B or C.

In certain preferred embodiments, the antisense strand of a nucleic acidmolecule (e.g., a siNA molecule) as disclosed herein as a component of apharmaceutical formulation includes a sequence corresponding to any oneof the antisense sequences shown on Table 5. In certain preferredembodiments the antisense strand and the sense strand are selected fromthe sequence pairs shown in Table 5. In some embodiments of a nucleicacid molecule (e.g., a siNA molecule) as disclosed herein includes theantisense and sense strands selected from the sequence pairs set forthin

SERPINH1_2 (SEQ ID NOS: 60 and 127),

SERPINH1_6 (SEQ ID NOS: 63 and 130),

SERPINH1_11 (SEQ ID NOS: 68 and 135),

SERPINH1_13 (SEQ ID NOS: 69 and 136),

SERPINH1_45 (SEQ ID NOS: 97 and 164),

SERPINH1_45a (SEQ ID NOS: 98 and 165),

SERPINH1_51 (SEQ ID NOS: 101 and 168),

SERPINH1_52 (SEQ ID NOS:102 and 169) or

SERPINH1_86 (SEQ ID NOS: 123 and 190).

In some preferred embodiments the antisense and sense strands areselected from the sequence pairs set forth in

SERPINH1_2 (SEQ ID NOS: 60 and 127),

SERPINH1_6 (SEQ ID NOS: 63 and 130),

SERPINH1_45a (SEQ ID NOS: 98 and 165), and

SERPINH1_51 (SEQ ID NOS: 101 and 168).

In some preferred embodiments of a nucleic acid molecule (e.g., a siNAmolecule) as disclosed herein as a component of a pharmaceuticalformulation includes the antisense and sense strands selected from thesequence pairs set forth in SERPINH1_2 (SEQ ID NOS: 60 and 127). In someembodiments the antisense and sense strands include the sequence pairsset forth in SERPINH1_6 (SEQ ID NOS: 63 and 130). In some embodiments ofa nucleic acid molecule (e.g., a siNA molecule) as disclosed hereinincludes the antisense and sense strands of the sequence pairs set forthin SERPINH1_11 (SEQ ID NOS: 68 and 135). In some embodiments theantisense and sense strands are the sequence pairs set forth inSERPINH1_13 (SEQ ID NOS: 69 and 136). In some embodiments the antisenseand sense strands are the sequence pairs set forth in SERPINH1_45 (SEQID NOS: 97 and 164). In some embodiments the antisense and sense strandsare the sequence pairs set forth in SERPINH1_45a (SEQ ID NOS: 98 and165). In some embodiments the antisense and sense strands are thesequence pairs set forth in SERPINH1_51 (SEQ ID NOS: 101 and 168).

In certain embodiments, the antisense strand of a nucleic acid molecule(e.g., a siNA molecule) as disclosed as a component of a pharmaceuticalformulation herein includes a sequence corresponding to any one of theantisense sequences shown on any one of Tables D or E.

In various embodiments of nucleic acid molecules (e.g., siNA molecules)as disclosed herein as a component of a pharmaceutical formulation, theantisense strand may be 15 to 49 nucleotides in length (e.g., 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotidesin length); or 17-35 nucleotides in length; or 17-30 nucleotides inlength; or 15-25 nucleotides in length; or 18-25 nucleotides in length;or 18-23 nucleotides in length; or 19-21 nucleotides in length; or 25-30nucleotides in length; or 26-28 nucleotides in length. In someembodiments of nucleic acid molecules (e.g., siNA molecules) asdisclosed herein, the antisense strand may be 19 nucleotides in length.Similarly, the sense strand of nucleic acid molecules (e.g., siNAmolecules) as disclosed herein may be 15 to 49 nucleotides in length(e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48or 49 nucleotides in length); or 17-35 nucleotides in length; or 17-30nucleotides in length; or 15-25 nucleotides in length; or 18-25nucleotides in length; or 18-23 nucleotides in length; or 19-21nucleotides in length; or 25-30 nucleotides in length; or 26-28nucleotides in length. In some embodiments of nucleic acid molecules(e.g., siNA molecules) as disclosed herein, the sense strand may be 19nucleotides in length. In some embodiments of nucleic acid molecules(e.g., siNA molecules) as disclosed herein, the antisense strand and thesense strand may be 19 nucleotides in length. The duplex region of thenucleic acid molecules (e.g., siNA molecules) as disclosed herein may be15-49 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48 or 49 nucleotides in length), 15-35nucleotides in length; or 15-30 nucleotides in length; or about 15-25nucleotides in length; or 17-25 nucleotides in length; or 17-23nucleotides in length; or 17-21 nucleotides in length; or 25-30nucleotides in length; or 25-28 nucleotides in length. In variousembodiments of nucleic acid molecules (e.g., siNA molecules) asdisclosed herein, the duplex region may be 19 nucleotides in length.

In certain embodiments, the sense and antisense strands of a nucleicacid (e.g., an siNA nucleic acid molecule) as provided herein as acomponent of a pharmaceutical formulation are separate polynucleotidestrands. In some embodiments, the separate antisense and sense strandsform a double-stranded structure via hydrogen bonding, for example,Watson-Crick base pairing. In some embodiments, the sense and antisensestrands are two separate strands that are covalently linked to eachother. In other embodiments, the sense and antisense strands are part ofa single polynucleotide strand having both a sense and antisense region;in some preferred embodiments the polynucleotide strand has a hairpinstructure.

In certain embodiments, the nucleic acid molecule (e.g., siNA molecule)is a double-stranded nucleic acid (dsNA) molecule that is symmetricalwith regard to overhangs, and has a blunt end on both ends. In otherembodiments, the nucleic acid molecule (e.g., siNA molecule) is a dsNAmolecule that is symmetrical with regard to overhangs, and has anoverhang on both ends of the dsNA molecule; preferably the molecule hasoverhangs of 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides; preferably themolecule has 2 nucleotide overhangs. In some embodiments, the overhangsare 5′ overhangs; in alternative embodiments, the overhangs are 3′overhangs. In certain embodiments, the overhang nucleotides are modifiedwith modifications as disclosed herein. In some embodiments, theoverhang nucleotides are 2′-deoxynucleotides.

In certain preferred embodiments, the nucleic acid molecule (e.g., siNAmolecule) as a component of a pharmaceutical formulation is a dsNAmolecule that is asymmetrical with regard to overhangs, and has a bluntend on one end of the molecule and an overhang on the other end of themolecule. In certain embodiments, the overhang is 1, 2, 3, 4, 5, 6, 7,or 8 nucleotides; preferably the overhang is 2 nucleotides. In somepreferred embodiments, an asymmetrical dsNA molecule has a 3′-overhang(for example a two nucleotide 3′-overhang) on one side of a duplexoccurring on the sense strand; and a blunt end on the other side of themolecule. In some preferred embodiments, an asymmetrical dsNA moleculehas a 5′-overhang (for example a two nucleotide 5′-overhang) on one sideof a duplex occurring on the sense strand; and a blunt end on the otherside of the molecule. In other preferred embodiments, an asymmetricaldsNA molecule has a 3′-overhang (for example a two nucleotide3′-overhang) on one side of a duplex occurring on the antisense strand;and a blunt end on the other side of the molecule. In some preferredembodiments, an asymmetrical dsNA molecule has a 5′-overhang (forexample a two nucleotide 5′-overhang) on one side of a duplex occurringon the antisense strand; and a blunt end on the other side of themolecule. In certain preferred embodiments, the overhangs are2′-deoxynucleotides.

In some embodiments, the nucleic acid molecule (e.g., siNA molecule) asa component of a pharmaceutical formulation has a hairpin structure(having the sense strand and antisense strand on one polynucleotide),with a loop structure on one end and a blunt end on the other end. Insome embodiments, the nucleic acid molecule has a hairpin structure,with a loop structure on one end and an overhang end on the other end(for example a 1, 2, 3, 4, 5, 6, 7, or 8 nucleotide overhang); incertain embodiments, the overhang is a 3′-overhang; in certainembodiments the overhang is a 5′-overhang; in certain embodiments theoverhang is on the sense strand; in certain embodiments the overhang ison the antisense strand.

In some preferred embodiments, the nucleic acid molecule is selectedfrom the nucleic acid molecules shown on Table 3.

The nucleic acid molecules (e.g., siNA molecule) disclosed herein as acomponent of a pharmaceutical formulation may include one or moremodifications or modified nucleotides such as described herein. Forexample, a nucleic acid molecule (e.g., siNA molecule) as providedherein may include a modified nucleotide having a modified sugar; amodified nucleotide having a modified nucleobase; or a modifiednucleotide having a modified phosphate group. Similarly, a nucleic acidmolecule (e.g., siNA molecule) as provided herein may include a modifiedphosphodiester backbone and/or may include a modified terminal phosphategroup.

Nucleic acid molecules (e.g., siNA molecules) as provided as a componentof a pharmaceutical formulation may have one or more nucleotides thatinclude a modified sugar moiety, for example as described herein. Insome preferred embodiments, the modified sugar moiety is selected fromthe group consisting of 2′OMe, 2′-methoxyethoxy, 2′-deoxy, 2′-fluoro,2′-allyl, 2′-O-(2-(methylamino)-2-oxoethyl), 4′-thio,4′-(CH₂)₂—O-2′-bridge, 2′-LNA (the ribose moiety of an LNA nucleotide ismodified with an extra bridge connecting the 2′ oxygen and 4′ carbon),and 2′-O—(N-methylcarbamate).

Nucleic acid molecules (e.g., siNA molecules) as provided as a componentof a pharmaceutical formulation may have one or more modifiednucleobase(s) for example as described herein, which preferably may beone selected from the group consisting of xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halo uracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substitutedadenines and guanines, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine, and acyclonucleotides.

Nucleic acid molecules (e.g., siNA molecules) as provided as a componentof a pharmaceutical formulation may have one or more modifications tothe phosphodiester backbone, for example as described herein. In somepreferred embodiments, the phosphodiester bond is modified bysubstituting the phosphodiester bond with a phosphorothioate, 3′-(or5′-) deoxy-3′-(or 5′-)thio-phosphorothioate, phosphorodithioate,phosphoroselenates, 3′-(or -5′)deoxy phosphinates, borano phosphates,3′-(or 5′-)deoxy-3′-(or 5′-)amino phosphoramidates, hydrogenphosphonates, borano phosphate esters, phosphoramidates, alkyl or arylphosphonates and phosphotriester or phosphorus linkages.

In various embodiments, the provided nucleic acid molecules (e.g., siNAmolecules) as a component of a pharmaceutical formulation may includeone or more modifications in the sense strand but not the antisensestrand. In some embodiments, the provided nucleic acid molecules (e.g.,siNA molecules) include one or more modifications in the antisensestrand but not the sense strand. In some embodiments, the providednucleic acid molecules (e.g., siNA molecules) include one or moremodifications in the both the sense strand and the antisense strand.

In some embodiments in which the provided nucleic acid molecules (e.g.,siNA molecules) as a component of a pharmaceutical formulation havemodifications, the sense strand includes a pattern of alternatingmodified and unmodified nucleotides, and/or the antisense strandincludes a pattern of alternating modified and unmodified nucleotides;in some preferred versions of such embodiments, the modification is a2′OMe moiety. The pattern of alternating modified and unmodifiednucleotides may start with a modified nucleotide at the 5′-end or 3′-endof one of the strands; for example the pattern of alternating modifiedand unmodified nucleotides may start with a modified nucleotide at the5′-end or 3′-end of the sense strand and/or the pattern of alternatingmodified and unmodified nucleotides may start with a modified nucleotideat the 5′-end or 3′-end of the antisense strand. When both the antisenseand sense strand include a pattern of alternating modified nucleotides,the pattern of modified nucleotides may be configured such that modifiednucleotides in the sense strand are opposite modified nucleotides in theantisense strand; or there may be a phase shift in the pattern such thatmodified nucleotides of the sense strand are opposite unmodifiednucleotides in the antisense strand and vice-versa.

The nucleic acid molecules (e.g., siNA molecules) as provided herein asa component of a pharmaceutical formulation may include one to three(i.e., 1, 2 or 3) deoxynucleotides at the 3′-end of the sense and/orantisense strand.

The nucleic acid molecules (e.g., siNA molecules) as provided herein asa component of a pharmaceutical formulation may include a phosphategroup at the 5′-end of the sense and/or antisense strand.

In one aspect, provided as a component of a pharmaceutical formulationare double-stranded nucleic acid molecules having the structure (A1):

(A1) 5′ (N)_(x)-Z 3′ (antisense strand) 3′ Z′-(N′)_(y)-z″ 5′ (sensestrand)wherein each of N and N′ is a nucleotide which may be unmodified ormodified, or an unconventional moiety;wherein each of (N)_(x) and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the next N or N′ by a covalent bond;wherein each of Z and Z′ is independently present or absent, but ifpresent independently includes 1-5 consecutive nucleotides ornon-nucleotide moieties or a combination thereof covalently attached atthe 3′-terminus of the strand in which it is present;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of (N′)_(y);wherein each of x and y is independently an integer between 18 and 40;wherein the sequence of (N′)_(y) has complementary to the sequence of(N)_(x); and wherein (N)_(x) includes an antisense sequence to SEQ IDNO:1.

In some embodiments, (N)_(x) includes an antisense oligonucleotidepresent in Table 4. In other embodiments, (N)_(x) is selected from anantisense oligonucleotide present in Tables B or C.

In some embodiments, the covalent bond joining each consecutive N or N′is a phosphodiester bond.

In some embodiments, x=y and each of x and y is 19, 20, 21, 22 or 23. Invarious embodiments, x=y=19.

In some embodiments of nucleic acid molecules (e.g., siNA molecules) asdisclosed herein, the double-stranded nucleic acid molecule is a siRNA,siNA or a miRNA.

In some embodiments, the antisense and sense strands are selected fromthe sequence pairs set forth in

SERPINH1_4 (SEQ ID NOS:195 and 220),

SERPINH1_12 (SEQ ID NOS:196 and 221),

SERPINH1_30 (SEQ ID NOS:199 and 224), and

SERPINH1_58 (SEQ ID NOS:208 and 233).

In some embodiments, the antisense and sense strands are the sequencepairs set forth in SERPINH1_4 (SEQ ID NOS:195 and 220). In someembodiments, the antisense and sense strands are the sequence pairs setforth in SERPINH1_12 (SEQ ID NOS:196 and 221). In some embodiments, theantisense and sense strands are the sequence pairs set forth inSERPINH1_30 (SEQ ID NOS:199 and 224). In some embodiments, the antisenseand sense strands are the sequence pairs set forth in SERPINH1_58 (SEQID NOS:208 and 233).

In some embodiments, the double-stranded nucleic acid molecules as acomponent of a pharmaceutical formulation comprise a DNA moiety or amismatch to the target at position 1 of the antisense strand(5′-terminus). Such a structure is described herein. According to oneembodiment provided are modified nucleic acid molecules having astructure (A2) set forth below:

(A2) 5′ N¹-(N)_(x)-Z 3′ (antisense strand) 3′ Z′-N²-(N′)_(y)-z″ 5′(sense strand)wherein each of N², N and N′ is an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of (N), and (N′)_(y) is an oligonucleotide in which eachconsecutive N or N′ is joined to the adjacent N or N′ by a covalentbond;wherein each of x and y is independently an integer between 17 and 39;wherein the sequence of (N′)_(y) has complementarity to the sequence of(N)_(x) and (N)_(x) has complementarity to a consecutive sequence in atarget RNA;wherein N¹ is covalently bound to (N)_(x) and is mismatched to thetarget RNA or is a complementary DNA moiety to the target RNA;wherein N¹ is a moiety selected from the group consisting of natural ormodified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine,adenosine or deoxyadenosine;wherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y); andwherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides, consecutivenon-nucleotide moieties or a combination thereof covalently attached atthe 3′-terminus of the strand in which it is present.

In some embodiments, the sequence of (N′)_(y) is fully complementary tothe sequence of (N)_(x). In various embodiments, sequence of N²—(N′)_(y)is complementary to the sequence of N¹—(N)_(x). In some embodiments,(N)_(x) comprises an antisense that is fully complementary to about 17to about 39 consecutive nucleotides in a target RNA. In otherembodiments, (N)_(x) comprises an antisense that is substantiallycomplementary to about 17 to about 39 consecutive nucleotides in atarget RNA.

In some embodiments, N¹ and N² form a Watson-Crick base pair. In someembodiments, N¹ and N² form a non-Watson-Crick base pair. In someembodiments, a base pair is formed between a ribonucleotide and adeoxyribonucleotide.

In some embodiments, x=y=18, x=y=19 or x=y=20. In preferred embodiments,x=y=18. When x=18 in N¹—(N)x, N¹ refers to position 1 and positions 2-19are included in (N)₁₈. When y=18 in N²—(N′)_(y), N² refers to position19 and positions 1-18 are included in (N′)₁₈.

In some embodiments, N¹ is covalently bound to (N)x and is mismatched tothe target RNA. In various embodiments, N¹ is covalently bound to(N)_(x) and is a DNA moiety complementary to the target RNA.

In some embodiments, a uridine in position 1 of the antisense strand issubstituted with an N¹ selected from adenosine, deoxyadenosine,deoxyuridine (dU), ribothymidine or deoxythymidine. In variousembodiments, N¹ is selected from adenosine, deoxyadenosine ordeoxyuridine.

In some embodiments, guanosine in position 1 of the antisense strand issubstituted with an N¹ selected from adenosine, deoxyadenosine, uridine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments,N¹ is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments, cytidine in position 1 of the antisense strand issubstituted with an N¹ selected from adenosine, deoxyadenosine, uridine,deoxyuridine, ribothymidine or deoxythymidine. In various embodiments,N¹ is selected from adenosine, deoxyadenosine, uridine or deoxyuridine.

In some embodiments, adenosine in position 1 of the antisense strand issubstituted with an N¹ selected from deoxyadenosine, deoxyuridine,ribothymidine or deoxythymidine. In various embodiments, N¹ selectedfrom deoxyadenosine or deoxyuridine.

In some embodiments, N¹ and N² form a base pair between uridine ordeoxyuridine, and adenosine or deoxyadenosine. In other embodiments, N¹and N² form a base pair between deoxyuridine and adenosine.

In some embodiments, the double-stranded nucleic acid molecule as acomponent of a pharmaceutical formulation is a siRNA, siNA or a miRNA.The double-stranded nucleic acid molecules as provided herein are alsoreferred to as “duplexes”.

In some embodiments (N)_(x) includes an antisense oligonucleotidepresent in Table 5. In some embodiments, x=y=18 and N¹—(N)_(x) includesan antisense oligonucleotide present in Table 4. In some embodimentsx=y=19 or x=y=20. In certain preferred embodiments, x=y=18. In someembodiments x=y−18 and the sequences of N¹—(N)_(x) and N²—(N′)_(y) areselected from the pair of oligonucleotides set forth in Table 4. In someembodiments, x=y=18 and the sequences of N¹—(N)_(x) and N²—(N′)_(y) areselected from the pair of oligonucleotides set forth in Tables D and E.In some embodiments, the antisense and sense strands are selected fromthe sequence pairs set forth in

SERPINH1_2 (SEQ ID NOS: 60 and 127),

SERPINH1_6 (SEQ ID NOS: 63 and 130),

SERPINH1_11 (SEQ ID NOS: 68 and 135),

SERPINH1_13 (SEQ ID NOS: 69 and 136),

SERPINH1_45 (SEQ ID NOS: 97 and 164),

SERPINH1_45a (SEQ ID NOS: 98 and 165),

SERPINH1_51 (SEQ ID NOS: 101 and 168),

SERPINH1_51a (SEQ ID NOS: 105 and 172),

SERPINH1_52 (SEQ ID NOS:102 and 169), and

SERPINH1_86 (SEQ ID NOS: 123 and 190).

In some preferred embodiments, the antisense and sense strands areselected from the sequence pairs set forth in

SERPINH1_2 (SEQ ID NOS: 60 and 127),

SERPINH1_6 (SEQ ID NOS: 63 and 130),

SERPINH1_45a (SEQ ID NOS: 98 and 165),

SERPINH1_51 (SEQ ID NOS: 101 and 168), and

SERPINH1_51a (SEQ ID NOS: 105 and 172).

In some preferred embodiments, the antisense and sense strands areselected from the sequence pairs set forth in SERPINH1_2 (SEQ ID NOS: 60and 127). In some embodiments, the antisense and sense strands are thesequence pairs set forth in SERPINH1_6 (SEQ ID NOS: 63 and 130). In someembodiments, the antisense and sense strands are the sequence pairs setforth in SERPINH1_11 (SEQ ID NOS: 68 and 135). In some embodiments, theantisense and sense strands are the sequence pairs set forth inSERPINH1_13 (SEQ ID NOS: 69 and 136). In some embodiments, the antisenseand sense strands are the sequence pairs set forth in SERPINH1_45 (SEQID NOS: 97 and 164). In some embodiments, the antisense and sensestrands are the sequence pairs set forth in SERPINH1_45a (SEQ ID NOS: 98and 165). In some embodiments, the antisense and sense strands are thesequence pairs set forth in SERPINH1_51 (SEQ ID NOS: 101 and 168). Insome embodiments, the antisense and sense strands are the sequence pairsset forth in SERPINH1_51a (SEQ ID NOS: 105 and 172). In someembodiments, the antisense and sense strands are the sequence pairs setforth in SERPINH1_52 (SEQ ID NOS:102 and 169). In some embodiments theantisense and sense strands are the sequence pairs set forth in (SEQ IDNOS: 123 and 190). In some preferred embodiments, the antisense andsense strands are selected from the sequence pairs set forth inSERPINH1_2 (SEQ ID NOS: 60 and 127), SERPINH1_6 (SEQ ID NOS: 63 and130), SERPINH1_45a (SEQ ID NOS: 98 and 165), SERPINH1_51 (SEQ ID NOS:101 and 168), and SERPINH1_51a (SEQ ID NOS: 105 and 172).

In some embodiments, N¹ and N² form a Watson-Crick base pair. In otherembodiments, N¹ and N² form a non-Watson-Crick base pair. In someembodiments N¹ is a modified riboadenosine or a modified ribouridine.

In some embodiments, N¹ and N² form a Watson-Crick base pair. In otherembodiments, N¹ and N² form a non-Watson-Crick base pair. In certainembodiments, N¹ is selected from the group consisting of riboadenosine,modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine.In other embodiments, N¹ is selected from the group consisting ofribouridine, deoxyribouridine, modified ribouridine, and modifieddeoxyribouridine.

In certain embodiments, position 1 in the antisense strand (5′-terminus)includes deoxyribouridine (dU) or adenosine. In some embodiments, N¹ isselected from the group consisting of riboadenosine, modifiedriboadenosine, deoxyriboadenosine, modified deoxyriboadenosine and N² isselected from the group consisting of ribouridine, deoxyribouridine,modified ribouridine, and modified deoxyribouridine. In certainembodiments, N¹ is selected from the group consisting of riboadenosineand modified riboadenosine and N² is selected from the group consistingof ribouridine and modified ribouridine.

In certain embodiments, N¹ is selected from the group consisting ofribouridine, deoxyribouridine, modified ribouridine, and modifieddeoxyribouridine and N² is selected from the group consisting ofriboadenosine, modified riboadenosine, deoxyriboadenosine, and modifieddeoxyriboadenosine. In certain embodiments, N¹ is selected from thegroup consisting of ribouridine and deoxyribouridine and N² is selectedfrom the group consisting of riboadenosine and modified riboadenosine.In certain embodiments, N¹ is ribouridine and N² is riboadenosine. Incertain embodiments, N¹ is deoxyribouridine and N² is riboadenosine.

In some embodiments of Structure (A2), N¹ includes 2′OMe modifiedribouracil or 2′OMe modified riboadenosine. In certain embodiments ofstructure (A2), N² includes a 2′OMe modified ribonucleotide ordeoxyribonucleotide.

In some embodiments of Structure (A2), N¹ includes 2′OMe modifiedribouracil or 2′OMe modified ribocytosine. In certain embodiments ofstructure (A2), N² includes a 2′OMe modified ribonucleotide.

In some embodiments each of N and N′ is an unmodified nucleotide. Insome embodiments, at least one of N or N′ includes a chemically modifiednucleotide or an unconventional moiety. In some embodiments, theunconventional moiety is selected from a mirror nucleotide, an abasicribose moiety and an abasic deoxyribose moiety. In some embodiments, theunconventional moiety is a mirror nucleotide, preferably an L-DNAmoiety. In some embodiments, at least one of N or N′ includes a 2′OMemodified ribonucleotide.

In some embodiments, the sequence of (N)_(y) is fully complementary tothe sequence of (N)_(x). In other embodiments, the sequence of (N′)_(y)is substantially complementary to the sequence of (N)_(x).

In some embodiments, (N)_(x) includes an antisense sequence that isfully complementary to about 17 to about 39 consecutive nucleotides in atarget mRNA. In other embodiments, (N)_(x) includes an antisense that issubstantially complementary to about 17 to about 39 consecutivenucleotides in a target mRNA.

In some embodiments of Structure A1 and Structure A2 the compound isblunt ended, for example, wherein both Z and Z′ are absent. In analternative embodiment, at least one of Z or Z′ is present. Z and Z′independently include one or more covalently linked modified and orunmodified nucleotides, including deoxyribonucleotides andribonucleotides, or an unconventional moiety, for example, invertedabasic deoxyribose moiety or abasic ribose moiety; a non-nucleotide C3,C4 or C5 moiety, an amino-6 moiety, a mirror nucleotide, and the like.In some embodiments, each of Z and Z′ independently includes a C3 moietyor an amino-C6 moiety. In some embodiments Z′ is absent and Z is presentand includes a non-nucleotide C3 moiety. In some embodiments Z is absentand Z′ is present and includes a non-nucleotide C3 moiety.

In some embodiments of Structure A1 and Structure A2, each N consists ofan unmodified ribonucleotide. In some embodiments of Structure A1 andStructure A2, each N′ consists of an unmodified nucleotide. In preferredembodiments, at least one of N and N′ is a modified ribonucleotide or anunconventional moiety.

In other embodiments, the compound of Structure A1 or Structure A2includes at least one ribonucleotide modified in the sugar residue. Insome embodiments, the compound includes a modification at the 2′position of the sugar residue. In some embodiments, the modification inthe 2′ position includes the presence of an amino, a fluoro, an alkoxyor an alkyl moiety. In certain embodiments, the 2′ modification includesan alkoxy moiety. In preferred embodiments, the alkoxy moiety is amethoxy moiety (2′OMe). In some embodiments, the nucleic acid compoundincludes 2′OMe modified alternating ribonucleotides in one or both ofthe antisense and the sense strands. In other embodiments, the compoundincludes 2′OMe modified ribonucleotides in the antisense strand, (N)_(x)or N¹—(N)_(x), only. In certain embodiments, the middle ribonucleotideof the antisense strand; e.g. ribonucleotide in position 10 in a 19-merstrand is unmodified. In various embodiments, the nucleic acid compoundincludes at least 5 alternating 2′OMe modified and unmodifiedribonucleotides. In additional embodiments, the compound of Structure A1or Structure A2 includes modified ribonucleotides in alternatingpositions wherein each ribonucleotide at the 5′ and 3′-termini of(N)_(x) or N¹—(N)_(x) are modified in their sugar residues, and eachribonucleotide at the 5′ and 3′-termini of (N′)_(y) or N²—(N)_(y) areunmodified in their sugar residues.

In some embodiments, the double-stranded molecule as a component of apharmaceutical formulation includes one or more of the followingmodifications

-   -   N in at least one of positions 5, 6, 7, 8, or 9 of the antisense        strand is selected from a 2′-5′-nucleotide or a mirror        nucleotide;    -   N′ in at least one of positions 9 or 10 of the sense strand is        selected from a 2′-5′-nucleotide and a pseudo-uridine; and    -   N′ in 4, 5, or 6 consecutive positions at the 3′-terminus        positions of (N′)_(y) comprises a 2′-5′-nucleotide.

In some embodiments, the double-stranded molecule includes a combinationof the following modifications

-   -   the antisense strand includes a 2′-5′-nucleotide or a mirror        nucleotide in at least one of positions 5, 6, 7, 8, or 9; and    -   the sense strand includes at least one of a 2′-5′-nucleotide and        a pseudo-uridine in positions 9 or 10.

In some embodiments, the double-stranded molecule includes a combinationof the following modifications

-   -   the antisense strand includes a 2′-5′-nucleotide or a mirror        nucleotide in at least one of positions 5, 6, 7, 8, or 9; and    -   the sense strand includes 4, 5, or 6 consecutive        2′-5′-nucleotides at the 3′-penultimate or 3′-terminal        positions.

In some embodiments, the sense strand ((N)_(x) or N¹—(N)_(x)) includes1, 2, 3, 4, 5, 6, 7, 8, or 9 2′OMe modified ribonucleotides. In someembodiments, the antisense strand includes 2′OMe modifiedribonucleotides at positions 2, 4, 6, 8, 11, 13, 15, 17 and 19. In otherembodiments, antisense strand includes 2′OMe modified ribonucleotides atpositions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In other embodiments,the antisense strand includes 2′OMe modified ribonucleotides atpositions 3, 5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments theantisense strand includes one or more 2′OMe modified pyrimidines. Insome embodiments, all the pyrimidine nucleotides in the antisense strandare 2′OMe modified. In some embodiments, the sense strand includes 2′OMemodified pyrimidines.

In some embodiments of Structure A1 and Structure A2, neither the sensestrand nor the antisense strand is phosphorylated at the 3′- and5′-termini. In other embodiments, one or both of the sense strand or theantisense strand are phosphorylated at the 3′-termini.

In some embodiments of Structure A1 and Structure A2, (N)_(y) includesat least one unconventional moiety selected from a mirror nucleotide, a2′-5′-nucleotide and a TNA. In some embodiments, the unconventionalmoiety is a mirror nucleotide. In various embodiments, the mirrornucleotide is selected from an L-ribonucleotide (L-RNA) and anL-deoxyribonucleotide (L-DNA). In preferred embodiments, the mirrornucleotide is L-DNA. In certain embodiments, the sense strand comprisesan unconventional moiety in position 9 or 10 (from the 5′-terminus). Inpreferred embodiments, the sense strand includes an unconventionalmoiety in position 9 (from the 5′-terminus). In some embodiments, thesense strand is 19 nucleotides in length and comprises 4, 5, or 6consecutive unconventional moieties in positions 15, (from the5′-terminus). In some embodiments, the sense strand includes 4consecutive 2′-5′-ribonucleotides in positions 15, 16, 17, and 18. Insome embodiments, the sense strand includes 5 consecutive2′-5′-ribonucleotides in positions 15, 16, 17, 18 and 19. In variousembodiments, the sense strand further comprises Z′. In some embodiments,Z′ includes a C3OH moiety or a C3Pi moiety.

In some embodiments, of Structure A1 (N′)_(y) includes at least oneL-DNA moiety. In some embodiments, x=y=19 and (N′)_(y), consists ofunmodified ribonucleotides at positions 1-17 and 19 and one L-DNA at the3′ penultimate position (position 18). In other embodiments, x=y=19 and(N′)_(y) consists of unmodified ribonucleotides at positions 1-16 and 19and two consecutive L-DNA at the 3′ penultimate position (positions 17and 18). In various embodiments, the unconventional moiety is anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate linkage. According to various embodiments, (N′)_(y) includes2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3′-terminus linkedby 2′-5′ internucleotide linkages. In one embodiment, four consecutivenucleotides at the 3′-terminus of (N¹)_(y) are joined by three 2′-5′phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides whichform the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl(3′OMe) sugar modification. Preferably the 3′-terminal nucleotide of(N′)_(y) includes a 2′OMe modification. In certain embodiments, x=y=19and (N′)_(y) includes two or more consecutive nucleotides at positions15, 16, 17, 18 and 19 include a nucleotide joined to an adjacentnucleotide by a 2′-5′ internucleotide bond (2′-5′ nucleotide). Invarious embodiments the nucleotide forming the 2′-5′ internucleotidebond includes a 3′ deoxyribose nucleotide or a 3′ methoxy nucleotide (3′H or 3′OMe in place of a 3′ OH). In some embodiments x=y=19 and (N′)_(y)includes 2′-5′ nucleotides at positions 15, 16 and 17 such that adjacentnucleotides are linked by a 2′-5′ internucleotide bond between positions15-16, 16-17 and 17-18; or at positions, 15, 16, 17, 18, and 19 suchthat adjacent nucleotides are linked by a 2′-5′ internucleotide bondbetween positions 15-16, 16-17, 17-18 and 18-19 and a 3′0H is availableat the 3′-terminal nucleotide or at positions 16, 17 and 18 such thatadjacent nucleotides are linked by a 2′-5′ internucleotide bond betweenpositions 16-17, 17-18 and 18-19. In some embodiments x=y=19 and(N′)_(y) includes 2′-5′-nucleotides at positions 16 and 17 or atpositions 17 and 18 or at positions 15 and 17 such that adjacentnucleotides are linked by a 2′-5′ internucleotide bond between positions16-17 and 17-18 or between positions 17-18 and 18-19 or betweenpositions 15-16 and 17-18, respectively. In other embodiments, thepyrimidine ribonucleotides (rU, rC) in (N′)_(y) are substituted withnucleotides joined to the adjacent nucleotide by a 2′-5′ internucleotidebond. In some embodiments, the antisense and sense strands are selectedfrom the sequence pairs set forth in SERPINH1_4, SERPINH1_12,SERPINH1_18, SERPINH1_30, SERPINH1_58 or SERPINH1_88, and x=y=19 and(N′)_(y) comprises five consecutive nucleotides at the 3′-terminusjoined by four 2′-5′ linkages, specifically the linkages between thenucleotides position 15-16, 16-17, 17-18 and 18-19.

In some embodiments, the linkages include phosphodiester bonds. In someembodiments, the antisense and sense strands are selected from thesequence pairs set forth in SERPINH1_4, SERPINH1_12, SERPINH1_18,SERPINH1_30, SERPINH1_58 or SERPINH1_88 and x=y=19 and (N′)_(y)comprises five consecutive nucleotides at the 3′-terminus joined by four2′-5′ linkages and optionally further includes Z′ and z′ independentlyselected from an inverted abasic moiety and a C3 alkyl [C3;1,3-propanediol mono(dihydrogen phosphate)] cap. The C3 alkyl cap iscovalently linked to the 3′ or 5′ terminal nucleotide. In someembodiments, the 3′ C3 terminal cap further comprises a 3′ phosphate. Insome embodiments, the 3′ C3 terminal cap further comprises a 3′-terminalhydroxy group.

In some embodiments, the antisense and sense strands as components of apharmaceutical formulation are selected from the sequence pairs setforth in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58or SERPINH1_88 and x=y=19 and (N′)_(y) includes an L-DNA position 18;and (N′)_(y) optionally further includes Z′ and z′ independentlyselected from an inverted abasic moiety and a C3 alkyl (C3;1,3-propanediol mono(dihydrogen phosphate)) cap.

In some embodiments, (N′)_(y) includes a 3′-terminal phosphate. In someembodiments, (N′)_(y) includes a 3′-terminal hydroxyl.

In some embodiments, the antisense and sense strands as components of apharmaceutical formulation are selected from the sequence pairs setforth in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58or SERPINH1_88 and x=y=19 and (N)_(x) includes 2′OMe modifiedribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or atpositions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments, theantisense and sense strands are selected from the sequence pairs setforth in SERPINH1_4, SERPINH1_12, SERPINH1_18, SERPINH1_30, SERPINH1_58and SERPINH1_88 and x=y=19 and (N), includes 2′OMe modified pyrimidines.In some embodiments, all pyrimidines in (N), include the 2′OMemodification.

In some embodiments, the antisense and sense strands are selected fromthe sequence pairs set forth in SERPINH1_2, SERPINH1_6, SERPINH1_11,SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN51a,SERPINH1_52 or SERPINH1_86 and x=y=18 and N² is a riboadenosine moiety.

In some embodiments, the antisense and sense strands are selected fromthe sequence pairs set forth in SERPINH1_2, SERPINH1_6, SERPINH1_11,SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPIN51a,SERPINH1_52 or SERPINH1_86 and x=y=18, and N²—(N′)_(y) includes fiveconsecutive nucleotides at the 3′-terminus joined by four 2′-5′linkages, specifically the linkages between the nucleotides position15-16, 16-17, 17-18 and 18-19. In some embodiments the linkages includephosphodiester bonds.

In some embodiments, the antisense and sense strands are selected fromthe sequence pairs set forth in SERPINH1_2, SERPINH1_6, SERPINH1_11,SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPINH1_51a,SERPINH1_52 or SERPINH1_86 and x=y=18 and N²—(N)_(y) includes fiveconsecutive nucleotides at the 3′-terminus joined by four 2′-5′ linkagesand optionally further includes Z′ and z′ independently selected from aninverted abasic moiety and a C3 alkyl [C3; 1,3-propanediolmono(dihydrogen phosphate)] cap.

In some embodiments, the antisense and sense strands are selected fromthe sequence pairs set forth in SERPINH1_2, SERPINH1_6, SERPINH1_11,SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPINH1_51a,SERPINH1_52 or SERPINH1_86 and x=y=18 and N²—(N)_(y) includes an L-DNAposition 18; and (N′)_(y) optionally further includes Z′ and z′independently selected from an inverted abasic moiety and a C3 alkyl[C3; 1,3-propanediol mono(dihydrogen phosphate)] cap.

In some embodiments, N²—(N′)_(y) comprises a 3′-terminal phosphate. Insome embodiments, N²—(N′)_(y) comprises a 3′-terminal hydroxyl.

In some embodiments, the antisense and sense strands as components of apharmaceutical formulation are selected from the sequence pairs setforth in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45,SERPINH1_45a, SERPINH1_51, SERPINH1_51a, SERPINH1_52 or SERPINH1_86 andx=y=18 and N¹—(N)_(x) includes 2′OMe modified ribonucleotides inpositions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or in positions 1, 3, 5, 9,11, 13, 15, 17, 19, or in positions 3, 5, 9, 11, 13, 15, 17, or inpositions 2, 4, 6, 8, 11, 13, 15, 17, 19. In some embodiments, theantisense and sense strands are selected from the sequence pairs setforth in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45,SERPINH1_45a, SERPINH1_51, SERPINH1_52 or SERPINH1_86 and x=y=18 andN¹—(N)_(x) includes 2′OMe modified ribonucleotides at positions 11, 13,15, 17 and 19. In some embodiments, the antisense and sense strands areselected from the sequence pairs set forth in SERPINH1_2, SERPINH1_6,SERPINH1_11, SERPINH1_13, SERPINH1-45, SERPINH1_45a, SERPINH1_51,SERPINH1_51a, SERPINH1_52 or SERPINH1_86 and x=y=18 and N¹—(N)_(x)includes 2′OMe modified ribonucleotides in positions 1, 3, 5, 7, 9, 11,13, 15, 17, 19 or in positions 3, 5, 7, 9, 11, 13, 15, 17, 19. In someembodiments, the antisense and sense strands are selected from thesequence pairs set forth in SERPINH1_2, SERPINH1_6, SERPINH1_11,SERPINH1_13, SERPINH1_45, SERPINH1_45a, SERPINH1_51, SERPINH1_52 orSERPINH1_86 and x=y=18 and N¹—(N)_(x) includes 2′OMe modifiedribonucleotides in positions positions 2, 4, 6, 8, 11, 13, 15, 17, 19.

In some embodiments, the antisense and sense strands as components of apharmaceutical formulation are selected from the sequence pairs setforth in SERPINH1_2, SERPINH1_6, SERPINH1_11, SERPINH1_13, SERPINH1_45,SERPINH1_45a, SERPINH1_51, SERPINH1_51a, SERPINH1_52 or SERPINH1_86 andx=y=18 and N¹—(N)^(x) includes 2′OMe modified pyrimidines. In someembodiments, all pyrimidines in (N), include the 2′OMe modification. Insome embodiments, the antisense strand further includes an L-DNA or a2′-5′ nucleotide in position 5, 6 or 7. In other embodiments, theantisense strand further includes a ribonucleotide which generates a2′-5′ internucleotide linkage in between the ribonucleotides inpositions 5-6 or 6-7.

In additional embodiments, N¹—(N)_(x) further includes Z wherein Zincludes a non-nucleotide overhang. In some embodiments thenon-nucleotide overhang is C3-C3[1,3-propanediol mono(dihydrogenphosphate)]2.

In some embodiments, of Structure A2, (N)_(y) includes at least oneL-DNA moiety. In some embodiments x=y=18 and (N′)_(y) consists ofunmodified ribonucleotides at positions 1-16 and 18 and one L-DNA at the3′ penultimate position (position 17). In other embodiments, x=y=18 and(N′)_(y) consists of unmodified ribonucleotides at position 1-15 and 18and two consecutive L-DNA at the 3′ penultimate position (positions 16and 17). In various embodiments, the unconventional moiety is anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate linkage. According to various embodiments, (N′)_(y) includes2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3′-terminus linkedby 2′-5′ internucleotide linkages. In one embodiment, four consecutivenucleotides at the 3′-terminus of (N′)_(y) are joined by three 2′-5′phosphodiester bonds, wherein one or more of the 2′-5′ nucleotides whichform the 2′-5′ phosphodiester bonds further includes a 3′-O-methyl(3′OMe) sugar modification. Preferably, the 3′-terminal nucleotide of(N′)_(y) includes a 2′OMe modification. In certain embodiments, x=y=18and in (N′)y two or more consecutive nucleotides at positions 14, 15,16, 17, and 18 include a nucleotide joined to an adjacent nucleotide bya 2′-5′ internucleotide bond. In various embodiments, the nucleotideforming the 2′-5′ internucleotide bond includes a 3′ deoxyribosenucleotide or a 3′ methoxy nucleotide. In some embodiments, x=y=18 and(N′)_(y) includes nucleotides joined to the adjacent nucleotide by a2′-5′ internucleotide bond between positions 15-16, 16-17 and 17-18 orbetween positions 16-17 and 17-18. In some embodiments, x=y=18 and(N′)_(y) includes nucleotides joined to the adjacent nucleotide by a2′-5′ internucleotide bond between positions 14-15, 15-16, 16-17, and17-18 or between positions 15-16, 16-17, and 17-18 or between positions16-17 and 17-18 or between positions 17-18 or between positions 15-16and 17-18. In other embodiments, the pyrimidine ribonucleotides (rU, rC)in (N′)_(y) are substituted with nucleotides joined to the adjacentnucleotide by a 2′-5′ internucleotide bond.

In some embodiments, the antisense and sense strands are selected fromthe oligonucleotide pairs set forth in Table 5 and identified herein asSERPINH1_2 (SEQ ID NOS: 60 and 127), SERPINH1_6 (SEQ ID NOS: 63 and130), SERPINH1_45a (SEQ ID NOS: 98 and 165), SERPINH1_51 (SEQ ID NOS:101 and 168), and SERPINH1_51a (SEQ ID NOS: 105 and 172).

In some embodiments, the double-stranded nucleic acid molecule as acomponent of a pharmaceutical formulation includes the antisense strandset forth in SEQ ID NO:127 and sense strand set forth in SEQ ID NO:60;identified herein as SERPINH1_2. In some embodiments the double-strandednucleic acid molecule has the structure

5′    UAUAGCACCCAUGUGUCUC -Z 3′ (antisense SEQ ID NO: 127)      ||||||||||||||||||| 3′ Z′-AUAUCGUGGGUACACAGAG - z″ 5′ (sense SEQID NO: 60)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y).

In some embodiments, provided is a double-stranded nucleic acid moleculewherein the antisense strand (SEQ ID NO:127) includes one or more 2′OMemodified pyrimidines and or purines, a 2′-5′-ribonucleotide in position5, 6, 7 or 8, and a 3′-terminal nucleotide or non-nucleotide overhang.In some embodiments, the sense strand (SEQ ID NO:60) includes 4 or 5consecutive 2′-5′-nucleotides at the 3′-terminal or penultimatepositions, a nucleotide or non-nucleotide moiety covalently attached atthe 3′-terminus and a cap moiety covalently attached at the 5′-terminus.In other embodiments, the sense strand (SEQ ID NO:60) includes one ormore 2′OMe pyrimidine, a nucleotide or non-nucleotide moiety covalentlyattached at the 3′-terminus and a cap moiety covalently attached at the5′-terminus.

In some embodiments, provided is a double-stranded nucleic acid moleculewherein the antisense strand (SEQ ID NO:127) includes 2′OMe modifiedribonucleotides at positions 1, 3, 5, 9, 11, 15, 17 and 19; a2′-5′-ribonucleotide at position 7; and a C3Pi-C3OH moiety covalentlyattached to the 3′-terminus; and the sense strand (SEQ ID NO:60) isselected from a sense strand which includes

-   -   2′-5′-ribonucleotides at positions 15, 16, 17, 18 and 19; a C3OH        3′-terminal non-nucleotide overhang; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   2′-5′-ribonucleotides at positions 15, 16, 17, 18 and 19; a        3′-terminal phosphate; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   2′OMe modified ribonucleotides at positions 5, 7, 13, and 16; a        2′-5′-ribonucleotide at position 18; a C3-OH moiety covalently        attached at the 3′-terminus; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   2′OMe modified ribonucleotides at positions 7, 13, 16 and 18; a        2′-5′-ribonucleotide at position 9; a C3OH moiety covalently        attached at the 3′-terminus; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   2′-5′-ribonucleotides at positions 15, 16, 17, 18, and 19; a        C3-Pi moiety covalently attached at the 3′-terminus; and an        inverted abasic deoxyribonucleotide moiety covalently attached        at the 5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:127) includes 2′OMe modified ribonucleotidesat positions 1, 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide atposition 7; and a C3Pi-C3OH moiety covalently attached to the3′-terminus; and the sense strand (SEQ ID NO:60) includes2′-5′-ribonucleotides at positions 15, 16, 17, 18, and 19; a C33′-terminal overhang; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:127) includes 2′OMe modified ribonucleotidesat positions 1, 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide atposition 7; and a C3Pi-C3OH 3′-terminal overhang; and the sense strand(SEQ ID NO:60) includes 2′-5′-ribonucleotides at positions 15, 16, 17,18, and 19; a 3′-terminal phosphate; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:127) includes 2′OMe modified ribonucleotidesat positions 1, 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide atposition 7 and a C3Pi-C3OH moiety covalently attached to the3′-terminus; and the sense strand (SEQ ID NO:60) includes 2′OMe modifiedribonucleotides at positions 5, 7, 13, and 16; a 2′-5′-ribonucleotide atposition 18; a C3-OH moiety covalently attached at the 3′-terminus; andan inverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:127) includes 2′OMe modified ribonucleotidesat positions 1, 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide atposition 7 and a C3Pi-C3OH moiety covalently attached to the3′-terminus; and the sense strand (SEQ ID NO:60) includes 2′OMe modifiedribonucleotides at positions 7, 13, 16 and 18; a 2′-5′-ribonucleotide atposition 9; a C3-OH moiety covalently attached at the 3′-terminus; andan inverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:127) includes 2′OMe modified ribonucleotidesat positions 1, 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide atposition 7; and a C3Pi-C3OH moiety covalently attached to the3′-terminus; and the sense strand (SEQ ID NO:60) includes2′-5′-ribonucleotides at positions 15, 16, 17, 18, and 19; a C3-Pimoiety covalently attached at the 3′-terminus; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the antisense strand (SEQ ID NO:127) includes 2′OMemodified ribonucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, 19;and a C3-C3 3′-terminal overhang; and the sense strand (SEQ ID NO:60)includes 2′OMe modified ribonucleotides at positions 7, 9, 13, 16 and18; and an inverted abasic deoxyribonucleotide moiety covalentlyattached at the 5′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:60) includes2′-5′-ribonucleotides at positions 15, 16, 17, 18, and 19; a 3′-terminalphosphate; and an inverted abasic deoxyribonucleotide moiety covalentlyattached at the 5′-terminus; and the antisense strand (SEQ ID NO:127)includes an antisense strand selected from one of

-   -   2′OMe modified ribonucleotides at positions 1, 3, 5, 7, 9, 11,        13, 15, 17, 19; and a C3Pi-C3OH moiety covalently attached to        the 3′-terminus; or    -   2′OMe modified ribonucleotides at positions 1, 3, 6, 8, 10, 12,        14, 17, 18; and a C3Pi-C3OH moiety covalently attached to the        3′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule as a component of a pharmaceutical formulation which includesthe antisense strand set forth in SEQ ID NO:130 and the sense strand setforth in SEQ ID NO:63; identified herein as

SERPINH1_6. In some embodiments the duplex comprises the structure

5′    UACUCGUCUCGCAUCUUGU -Z 3′ (antisense SEQ ID NO: 130)      ||||||||||||||||||| 3′ Z′-AUGAGCAGAGCGUAGAACA -z″ 5′ (sense SEQ IDNO: 63)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y).

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:63) includes one or more2′OMe modified pyrimidines; a 3′-terminal nucleotide or non-nucleotideoverhang; and cap moiety covalently attached at the 5′-terminus. In someembodiments, the antisense strand (SEQ ID NO:130) includes one or more2′OMe modified pyrimidine; a nucleotide or non-nucleotide moietycovalently attached at the 3′-terminus; and a cap moiety covalentlyattached at the 5′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe modifiedribonucleotides at positions 2, 14 and 18; a C3OH or C3Pi moietycovalently attached at the 3′-terminus; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand (SEQ ID NO:130) is selected from an antisensestrand that includes

-   -   2′OMe modified ribonucleotides in positions 1, 3, 5, 9, 11, 13,        15 and 17; a 2′-5′-ribonucleotide at position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus; or    -   2′OMe modified ribonucleotides in positions 1, 3, 5, 7, 9, 12,        13 and 17; a 2′-5′-ribonucleotide at position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus; or    -   2′OMe modified ribonucleotides in positions 3, 5, 9, 11, 13, 15        and 17; a 2′-5′-ribonucleotide at position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus; or    -   2′OMe modified ribonucleotides in positions 3, 5, 9, 11, 13, 15        and 17; a dU in position 1; a 2′-5′-ribonucleotide in position        7; and a C3Pi-C3OH moiety covalently attached to the        3′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:63) includes 2′OMe modifiedribonucleotides in positions 2, 14 and 18; a C3-OH moiety covalentlyattached at the 3′-terminus; and an inverted abasic deoxyribonucleotidemoiety covalently attached at the 5′-terminus; and the antisense strand(SEQ ID NO:130) includes 2′OMe modified ribonucleotides in positions 1,3, 5, 9, 11, 13, 15 and 17; a 2′-5′-ribonucleotide in position 7; and aC3Pi-C3OH moiety covalently attached to the 3′-terminus.

In some embodiments provided herein is a duplex oligonucleotide moleculewherein the sense strand (SEQ ID NO:63) includes 2′OMe modifiedribonucleotides in positions 14 and 18 and optionally in position 2; aC3-OH moiety covalently attached at the 3′-terminus; and an invertedabasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:130) includes 2′OMemodified ribonucleotides in positions 1, 3, 5, 7, 9, 12, 13, and 17; a2′-5′-ribonucleotide at position 7; and a C3Pi-C3OH moiety covalentlyattached to the 3′-terminus.

In some embodiments provided herein is a duplex oligonucleotide moleculewherein the sense strand (SEQ ID NO:63) includes 2′OMe modifiedribonucleotides in positions 14 and 18; a C3-OH moiety covalentlyattached at the 3′-terminus; and an inverted abasic deoxyribonucleotidemoiety covalently attached at the 5′-terminus; and the antisense strand(SEQ ID NO:130) is selected from an antisense strand which includes

-   -   2′OMe modified ribonucleotides in positions 1, 3, 5, 9, 11, 13,        15 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3Pi        or C3Pi-C3OH moiety covalently attached to the 3′-terminus; or    -   2′OMe modified ribonucleotides in positions 1, 3, 5, 7, 9, 12,        13, and 17; a 2′-5′-ribonucleotide in position 7; and a        C3Pi-C3Pi or C3Pi-C3OH moiety covalently attached to the        3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:63) includes 2′OMe modified ribonucleotides inpositions 14 and 18; a C3-OH moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:130) includes 2′OMe modified ribonucleotides in positions 1, 3, 5, 9,11, 13, 15 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OHmoiety covalently attached to the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:63) includes 2′OMe modified ribonucleotides inpositions 14 and 18; a C3-OH moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:130) includes 2′OMe modified ribonucleotides in positions 1, 3, 5, 7,9, 12, 13, and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH3′-terminal overhang.

In some embodiments, the duplex s a component of a pharmaceuticalformulation includes the antisense strand set forth in SEQ ID NO:165 andsense strand set forth in SEQ ID NO:98; identified herein asSERPINH1_45a. In some embodiments, the duplex comprises the structure

5′    AGGAAGUUGAUCUUGGAGU -Z 3′ (antisense SEQ ID NO: 165)      ||||||||||||||||||| 3′ Z′-UCCUUCAACUAGAACCUCA -z″ 5′ (sense SEQ IDNO: 98)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y)

In some embodiments, the sense strand (SEQ ID NO:98) includes2′-5′-ribonucleotides in positions 15, 16, 17, and 18 or 15, 16, 17, 18,and 19; a nucleotide or non-nucleotide moiety covalently attached at the3′-terminus; and a cap moiety covalently attached at the 5′-terminus. Insome embodiments the antisense strand (SEQ ID NO:165) includes 2′OMemodified pyrimidine and or purines; a 2′-5′ nucleotide in position 5, 6,7, or 8; and a nucleotide or non-nucleotide moiety covalently attachedat the 3′-terminus.

In some embodiments, the sense strand (SEQ ID NO:98) includes2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; a C3Pi orC3-OH 3′-terminal non-nucleotide moiety; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand (SEQ ID NO:165) includes an antisense strandselected from one of

-   -   2′OMe modified ribonucleotides in positions 2, 4, 6, 8, 11, 13,        15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a        C3Pi-C3Pi or C3Pi-C3OH 3′-terminal overhang; or    -   2′OMe modified ribonucleotides in positions 2, 4, 6, 8, 11, 13,        15, 17 and 19; and a C3Pi-C3Pi or C3Pi-C3OH 3′-terminal        overhang;    -   2′OMe modified ribonucleotides in positions 1, 3, 5, 9, 11, 13,        15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a        C3Pi-C3Pi or C3Pi-C3OH 3′-terminal overhang; or    -   2′OMe modified ribonucleotides in positions 1, 3, 5, 7, 9, 11,        13, 15, 17 and 19; and a C3Pi-C3Pi or C3Pi-C3OH 3′-terminal        overhang.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:98) includes 2′-5′-ribonucleotides in positions15, 16, 17, 18, and 19; a C3-OH 3′-terminal moiety; and an invertedabasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:165) includes 2′OMemodified ribonucleotides in positions 2, 4, 6, 8, 11, 13, 15, 17, and19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-COH 3′-terminaloverhang.

In some embodiments, the double-stranded nucleic acid molecule s acomponent of a pharmaceutical formulation includes the antisense strandset forth in SEQ ID NO:168 and sense strand set forth in SEQ ID NO:101;identified herein as SERPINH1_51. In some embodiments, the duplexcomprises the structure

5′    UCACCCAUGUGUCUCAGGA -Z 3′ (antisense SEQ ID NO: 168)      ||||||||||||||||||| 3′ Z′-AGUGGGUACACAGAGUCCU -z″ 5′ (sense SEQ IDNO: 101)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y).

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:101) includes 2′OMemodified pyrimidines, optionally a 2′-5′-ribonucleotide in position 9 or10; a nucleotide or non-nucleotide moiety covalently attached at the3′-terminus; and optionally a cap moiety covalently attached at the5′-terminus. In some embodiments the antisense strand (SEQ ID NO:168)includes 2′OMe modified pyrimidine and or purines; a 2′-5′ nucleotide inposition 5, 6, 7, or 8; and a nucleotide or non-nucleotide moietycovalently attached at the 3′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:101) includes 2′OMemodified pyrimidines in positions 4, 11, 13, and 17; optionally a2′-5′-ribonucleotide in position 9 or 10; a C3Pi or C3OH non-nucleotidemoiety covalently attached at the 3′-terminus; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand (SEQ ID NO:168) is selected from an antisensestrand which includes

-   -   a) 2′OMe modified ribonucleotides in positions 1, 8, and 15; a        2′-5′-ribonucleotide in position 6 or 7; and a C3Pi-C3OH        overhang covalently attached at the 3′-terminus; or    -   b) 2′OMe modified ribonucleotides in positions 1, 4, 8, 13 and        15; a 2′-5′-ribonucleotide in position 6 or 7; and a C3Pi-C3OH        overhang covalently attached at the 3′-terminus; or    -   c) 2′OMe modified ribonucleotides in positions 1, 4, 8, 11 and        15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH        overhang covalently attached at the 3′-terminus; or    -   d) 2′OMe modified ribonucleotides in positions 1, 3, 8, 12, 13,        and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH        moiety covalently attached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:101) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; optionally a 2′-5′-ribonucleotide inposition 9; a C3-OH non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:168) includes 2′OMe modified ribonucleotides in positions 1, 8, and15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moietycovalently attached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:101) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; optionally a 2′-5′-ribonucleotide inposition 9; a C3-OH non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:168) includes 2′OMe modified ribonucleotides in positions 1, 4, 8, 13and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moietycovalently attached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:101) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; a 2′-5′-ribonucleotide in position 9; aC3OH non-nucleotide moiety covalently attached at the 3′-terminus; andan inverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:168) includes 2′OMemodified ribonucleotides in positions 1, 4, 8, 11 and 15; a2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalentlyattached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:101) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; a 2′-5′-ribonucleotide in position 9; aC3OH non-nucleotide moiety covalently attached at the 3′-terminus; andan inverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:168) includes 2′OMemodified ribonucleotides in positions 1, 3, 8, 12, 13, and 15; a2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalentlyattached at the 3′-terminus.

In some embodiments, the double-stranded nucleic acid molecule is acomponent of a pharmaceutical formulation includes the antisense strandset forth in SEQ ID NO:168 and sense strand set forth in SEQ ID NO:101;identified herein as SERPINH1_51a. In some embodiments the duplexcomprises the structure

5′    ACACCCAUGUGUCUCAGGA-Z 3′ (antisense SEQ ID NO: 172)   ||||||||||||||||||| 3′ Z′-UGUGGGUACACAGAGUCCU-z″ 5′ (sense SEQ ID NO:105)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y).

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:105) includes 2′OMemodified pyrimidines; optionally a 2′-5′-ribonucleotide in position 9 or10; a nucleotide or non-nucleotide moiety covalently attached at the3′-terminus; and optionally a cap moiety covalently attached at the5′-terminus. In some embodiments the antisense strand (SEQ ID NO:172)includes 2′OMe modified pyrimidine and or purines; a 2′-5′ nucleotide inposition 5, 6, 7, or 8; and a nucleotide or non-nucleotide moietycovalently attached at the 3′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:105) includes 2′OMemodified pyrimidines in positions 4, 11, 13, and 17; optionally a2′-5′-ribonucleotide in position 9 or 10; a C3Pi or C3OH non-nucleotidemoiety covalently attached at the 3′-terminus; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand (SEQ ID NO:172) is selected from an antisensestrand which includes

-   -   a) 2′OMe modified ribonucleotides in positions 8, and 15; a        2′-5′-ribonucleotide in position 6 or 7; and a C3Pi-C3OH moiety        covalently attached at the 3′-terminus; or    -   b) 2′OMe modified ribonucleotides in positions 4, 8, 13 and 15;        a 2′-5′-ribonucleotide in position 6 or 7; and a C3Pi-C3OH        moiety covalently attached at the 3′-terminus; or    -   c) 2′OMe modified ribonucleotides in positions 4, 8, 11 and 15;        a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety        covalently attached at the 3′-terminus; or    -   d) 2′OMe modified ribonucleotides in positions 3, 8, 12, 13, and        15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety        covalently attached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:105) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; optionally a 2′-5′-ribonucleotide inposition 9; a C3-OH non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:172) includes 2′OMe modified ribonucleotides in positions 8 and 15; a2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalentlyattached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:105) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; optionally a 2′-5′-ribonucleotide inposition 9; a C3-OH non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:172) includes 2′OMe modified ribonucleotides in positions 4, 8, 13and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moietycovalently attached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:105) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; a 2′-5′-ribonucleotide in position 9; aC3-OH non-nucleotide moiety covalently attached at the 3′-terminus; andan inverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:172) includes 2′OMemodified ribonucleotides in positions 4, 8, 11 and 15; a2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalentlyattached at the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:105) includes 2′OMe modified ribonucleotides inpositions 4, 11, 13, and 17; a 2′-5′-ribonucleotide in position 9; aC3OH non-nucleotide moiety covalently attached at the 3′-terminus; andan inverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:172) includes 2′OMemodified ribonucleotides in positions 3, 8, 12, 13, and 15; a2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalentlyattached at the 3′-terminus.

In some embodiments, the antisense and sense strands are selected fromthe oligonucleotide pairs set forth in Table 5 and identified herein asSERPINH1_4 (SEQ ID NOS: 195 and 220) and SERPINH1_12 (SEQ ID NOS: 196and 221).

In some embodiments, the double-stranded nucleic acid molecule is acomponent of a pharmaceutical formulation includes the antisense strandset forth in SEQ ID NO:220 and sense strand set forth in SEQ ID NO:195;identified herein as SERPINH1_4. In some embodiments, thedouble-stranded nucleic acid molecule has the structure

5′    AAUAGCACCCAUGUGUCUC-Z 3′ (antisense SEQ ID NO: 220)   ||||||||||||||||||| 3′ Z′-UUAUCGUGGGUACACAGAG-z″ 5′ (sense SEQ ID NO:195)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y).

In some embodiments provided is a double-stranded nucleic acid moleculewherein the antisense strand (SEQ ID NO:220) includes 2′OMe modifiedribonucleotides in positions 3, 5, 9, 11, 15, 17 and 19, a2′-5′-ribonucleotide in position 7, and a C3Pi-C3OH moiety covalentlyattached to the 3′-terminus; and the sense strand (SEQ ID NO:195) isselected from a sense strand which includes

-   -   a) 2′-5′-ribonucleotides in positions 15, 16, 17, 18 and 19, a        C3OH moiety covalently attached to the 3′-terminus; and an        inverted abasic deoxyribonucleotide moiety covalently attached        at the 5′-terminus; or    -   b) 2′-5′-ribonucleotides in positions 15, 16, 17, 18 and 19, a        3′-terminal phosphate; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   c) 2′OMe modified ribonucleotides in positions 5, 7, 13, and 16;        a 2′-5′-ribonucleotide in position 18; a C3OH moiety covalently        attached at the 3′-terminus; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   d) 2′OMe modified ribonucleotides in positions 7, 13, 16 and 18;        a 2′-5′-ribonucleotide in position 9; a C3OH moiety covalently        attached at the 3′-terminus; and an inverted abasic        deoxyribonucleotide moiety covalently attached at the        5′-terminus; or    -   e) 2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; a        C3Pi moiety covalently attached at the 3′-terminus; and an        inverted abasic deoxyribonucleotide moiety covalently attached        at the 5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:220) includes 2′OMe modified ribonucleotidesin positions 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide in position7; and a C3Pi-C3OH moiety covalently attached to the 3′-terminus; andthe sense strand (SEQ ID NO:195) includes 2′-5′-ribonucleotides inpositions 15, 16, 17, 18, and 19; a C3 moiety covalently attached to the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:220) includes 2′OMe modified ribonucleotidesin positions 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide in position7; and a C3Pi-C3OH moiety covalently attached to the 3′-terminus; andthe sense strand (SEQ ID NO:195) includes 2′-5′-ribonucleotides inpositions 15, 16, 17, 18, and 19; a 3′-terminal phosphate; and aninverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:220) includes 2′OMe modified ribonucleotidesin positions 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide in position7; and a C3Pi-C3OH moiety covalently attached to the 3′-terminus; andthe sense strand (SEQ ID NO:195) includes 2′OMe modified ribonucleotidesin positions 5, 7, 13, and 16; a 2′-5′-ribonucleotide in position 18; aC3OH moiety covalently attached at the 3′-terminus; and an invertedabasic deoxyribonucleotide moiety covalently attached at the5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:220) includes 2′OMe modified ribonucleotidesin positions 3, 5, 9, 11, 15, 17, 19; a 2′-5′-ribonucleotide in position7; and a C3Pi-C3OH moiety covalently attached to the 3′-terminus; andthe sense strand (SEQ ID NO:195) includes 2′OMe modified ribonucleotidesin positions 7, 13, 16 and 18; a 2′-5′-ribonucleotide in position 9; aC3OH moiety covalently attached at the 3′-terminus; and an invertedabasic deoxyribonucleotide moiety covalently attached at the5′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein theantisense strand (SEQ ID NO:220) includes 2′OMe modified ribonucleotidesin positions 3, 5, 9, 11, 15, 17, 19; a T-5′-ribonucleotide in position7; and a C3Pi-C3OH moiety covalently attached to the 3′-terminus; andthe sense strand (SEQ ID NO:195) includes T-5′-ribonucleotides inpositions 15, 16, 17, 18, and 19; a C3Pi moiety covalently attached atthe 3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the antisense strand (SEQ ID NO:220) includes 2′OMemodified ribonucleotides in positions 1, 3, 5, 9, 11, 13, 15, 17, 19;and a C3Pi-C3OH moiety covalently attached to the 3′-terminus; and thesense strand (SEQ ID NO:195) includes 2′OMe modified ribonucleotides inpositions 7, 9, 13, 16 and 18; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus.

In some embodiments provided herein is a double-stranded nucleic acidmolecule wherein the sense strand (SEQ ID NO:195) includes2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; a 3′-terminalphosphate and an inverted abasic deoxyribonucleotide moiety covalentlyattached at the 5′-terminus; and the antisense strand (SEQ ID NO:220)includes an antisense strand selected from one of

-   -   a) 2′OMe modified ribonucleotides in positions 3, 5, 7, 9, 11,        13, 15, 17, 19; and a C3Pi-C3OH moiety covalently attached to        the 3′-terminus; or    -   b) 2′OMe modified ribonucleotides in positions 1, 3, 6, 8, 10,        12, 14, 17, 18; and a C3Pi-C3OH moiety covalently attached to        the 3′-terminus.

In some embodiments, provided is a double-stranded nucleic acid moleculeis a component of a pharmaceutical formulation which includes theantisense strand set forth in SEQ ID NO:130 and the sense strand setforth in SEQ ID NO:63; identified herein as SERPINH1_12. In someembodiments the duplex comprises the structure

5′    AACUCGUCUCGCAUCUUGU-Z 3′ (antisense SEQ ID NO: 221)   ||||||||||||||||||| 3′ Z′-UUGAGCAGAGCGUAGAACA-z″ 5′ (sense SEQ ID NO:196)wherein each “|” represents base pairing between the ribonucleotides;wherein each of A, C, G, U is independently an unmodified or modifiedribonucleotide, or an unconventional moiety;wherein each of Z and Z′ is independently present or absent, but ifpresent is independently 1-5 consecutive nucleotides or non-nucleotidemoieties or a combination thereof covalently attached at the 3′-terminusof the strand in which it is present; andwherein z″ may be present or absent, but if present is a capping moietycovalently attached at the 5′-terminus of N²—(N′)_(y).

In some embodiments, provided is a double-stranded nucleic acid moleculewherein the sense strand (SEQ ID NO:196) includes one or more 2′OMemodified pyrimidines; a 3′-terminal nucleotide or non-nucleotideoverhang; and a cap moiety covalently attached at the 5′-terminus. Insome embodiments, the antisense strand (SEQ ID NO:221) includes one ormore 2′OMe modified pyrimidines; a nucleotide or non-nucleotide moietycovalently attached at the 3′-terminus; and a cap moiety covalentlyattached at the 5′-terminus.

In some embodiments provided is a double-stranded nucleic acid moleculewherein the sense strand (SEQ ID NO:196) includes 2′OMe modifiedribonucleotides in positions 2, 14 and 18; a C3OH moiety covalentlyattached at the 3′-terminus; and an inverted abasic deoxyribonucleotidemoiety covalently attached at the 5′-terminus; and the antisense strand(SEQ ID NO:221) is selected from an antisense strand which includes

-   -   a) 2′OMe modified ribonucleotides in positions 3, 5, 9, 11, 13,        15 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus; or    -   b) 2′OMe modified ribonucleotides in positions 3, 5, 7, 9, 12,        13 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus.

In some embodiments provided is a double-stranded nucleic acid moleculewherein the sense strand (SEQ ID NO:196) includes 2′OMe modifiedribonucleotides in positions 2, 14 and 18; a C3-OH moiety covalentlyattached at the 3′-terminus; and an inverted abasic deoxyribonucleotidemoiety covalently attached at the 5′-terminus; and the antisense strand(SEQ ID NO:221) includes 2′OMe modified ribonucleotides in positions 3,5, 9, 11, 13, 15 and 17; a 2′-5′-ribonucleotide in position 7; and aC3Pi-C3OH moiety covalently attached to the 3′-terminus.

In some embodiments provided is a duplex oligonucleotide moleculewherein the sense strand (SEQ ID NO:196) includes 2′OMe modifiedribonucleotides in positions 14 and 18 and optionally in position 2; aC3-OH moiety covalently attached at the 3′-terminus; and an invertedabasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand (SEQ ID NO:221) includes 2′OMemodified ribonucleotides in positions 3, 5, 7, 9, 12, 13, and 17; a2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH moiety covalentlyattached to the 3′-terminus.

In some embodiments provided is a duplex oligonucleotide moleculewherein the sense strand (SEQ ID NO:196) includes 2′OMe modifiedribonucleotides in positions 14 and 18; a C3-OH moiety covalentlyattached at the 3′-terminus; and an inverted abasic deoxyribonucleotidemoiety covalently attached at the 5′-terminus; and the antisense strand(SEQ ID NO:221) is selected from an antisense strand which includes

-   -   a) 2′OMe modified ribonucleotides in positions 3, 5, 9, 11, 13,        15 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus; or    -   b) 2′OMe modified ribonucleotides in positions 3, 5, 7, 9, 12,        13 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH        moiety covalently attached to the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:196) includes 2′OMe modified ribonucleotides inpositions 14 and 18; a C3-OH moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:221) includes 2′OMe modified ribonucleotides in positions 1, 3, 5, 9,11, 13, 15 and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OHmoiety covalently attached to the 3′-terminus.

Provided herein is a double-stranded nucleic acid molecule wherein thesense strand (SEQ ID NO:196) includes 2′OMe modified ribonucleotides inpositions 14 and 18; a C3-OH moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand (SEQ IDNO:221) includes 2′OMe modified ribonucleotides in positions 1, 3, 5, 7,9, 12, 13, and 17; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OHmoiety covalently attached to the 3′-terminus.

In further embodiments of Structures A1 and A2, (N′)y includes 1-8modified ribonucleotides wherein the modified ribonucleotide is a DNAnucleotide. In certain embodiments (N′)_(y) includes 1, 2, 3, 4, 5, 6,7, or up to 8 DNA moieties.

In some embodiments, either Z or Z′ is present and independentlyincludes two non-nucleotide moieties.

In additional embodiments, Z and Z′ are present and each independentlyincludes two non-nucleotide moieties.

In some embodiments, each of Z and Z′ includes an abasic moiety, forexample a deoxyribo-abasic moiety (referred to herein as “dAb”) orribo-abasic moiety (referred to herein as “rAb”). In some embodimentseach of Z and/or Z′ includes two covalently linked abasic moieties andis for example dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb, wherein eachmoiety is covalently attached to an adjacent moiety, preferably via aphospho-based bond. In some embodiments, the phospho-based bond includesa phosphorothioate, a phosphonoacetate or a phosphodiester bond. Inpreferred embodiments, the phospho-based bond includes a phosphodiesterbond.

In some embodiments, each of Z and/or Z′ independently includes an alkylmoiety, optionally propane ((CH₂)₃) moiety (“C3”) or a derivativethereof including propanediol (C3OH) and phospho derivative ofpropanediol (“C3Pi”). In some embodiments, each of Z and/or Z′ includestwo alkyl moieties covalently linked to the 3′-terminus of the antisensestrand or sense strand via a phosphodiester or phosphorothioate linkageand covalently linked to one another via a phosphodiester orphosphorothioate linkage and in some examples is C3Pi-C3Pi or C3Pi-C3OH.The 3′-terminus of the antisense strand and/or the 3′-terminus of thesense strand is covalently attached to a C3 moiety via a phospho-basedbond and the C3 moiety is covalently conjugated to a C3OH moiety via aphospho-based bond. In some embodiments the phospho-based bonds includea phosphorothioate, a phosphonoacetate or a phosphodiester bond. Inpreferred embodiments, the phospho-based bond includes a phosphodiesterbond.

In various embodiments of Structure A1 or Structure A2, Z and Z′ areabsent. In other embodiments Z or Z′ is present. In some embodiments,each of Z and/or Z′ independently includes a C2, C3, C4, C5 or C6 alkylmoiety, optionally a C3 moiety or a derivative thereof includingpropanol (C3OH/C3OH), propanediol, and phosphodiester derivative ofpropanediol (C3Pi). In preferred embodiments, each of Z and/or Z′includes two hydrocarbon moieties and in some examples is C3Pi-C3OH orC3Pi-C3Pi. Each C3 is covalently conjugated to an adjacent C3 via acovalent bond, preferably a phospho-based bond. In some embodiments, thephospho-based bond is a phosphorothioate, a phosphonoacetate or aphosphodiester bond.

In specific embodiments, x=y=19 and Z comprises at least one C3 alkyloverhang. In some embodiments the C3-C3 overhang is covalently attachedto the 3′-terminus of (N)_(x) or (N′)_(y) via a covalent linkage,preferably a phosphodiester linkage. In some embodiments, the linkagebetween a first C3 and a second C3 is a phosphodiester linkage. In someembodiments, the 3′ non-nucleotide overhang is C3Pi-C3Pi. In someembodiments the 3′ non-nucleotide overhang is C3Pi-C3Pi. In someembodiments, the 3′ non-nucleotide overhang is C3Pi-C3OH.

In various embodiments, the alkyl moiety comprises an alkyl derivativeincluding a C3 alkyl, C4 alkyl, C5 alkyl or C6 alkyl moiety comprising aterminal hydroxyl, a terminal amino, or terminal phosphate group. Insome embodiments, the alkyl moiety is a C3 alkyl or C3 alkyl derivativemoiety. In some embodiments, the C3 alkyl moiety comprises propanol,propyl phosphate, propyl phosphorothioate or a combination thereof. TheC3 alkyl moiety is covalently linked to the 3′-terminus of (N′)_(y)and/or the 3′-terminus of (N)_(x) via a phosphodiester bond. In someembodiments, the alkyl moiety comprises propanol, propyl phosphate, orpropyl phosphorothioate. In some embodiments, each of Z and Z′ isindependently selected from propanol, propyl phosphate, propylphosphorothioate, combinations thereof, or multiples thereof, inparticular 2 or 3 covalently linked propanol, propyl phosphate, propylphosphorothioate, or combinations thereof. In some embodiments, each ofZ and Z′ is independently selected from propyl phosphate, propylphosphorothioate, propyl phospho-propanol; propyl phospho-propylphosphorothioate; propyl phospho-propyl phosphate; (propyl phosphate)₃,(propyl phosphate)₂-propanol, (propyl phosphate)₂-propylphosphorothioate. Any propane or propanol conjugated moiety can beincluded in Z or Z′.

The structures of exemplary 3′-terminal non-nucleotide moieties are asfollows:

In some embodiments, each of Z and Z′ is independently selected frompropanol, propyl phosphate, propyl phosphorothioate, combinationsthereof or multiples thereof.

In some embodiments, each of Z and Z′ is independently selected frompropyl phosphate, propyl phosphorothioate, propyl phospho-propanol;propyl phospho-propyl phosphorothioate; propylphospho-propyl phosphate;(propyl phosphate)₃, (propyl phosphate)₂-propanol, (propylphosphate)₂-propyl phosphorothioate. Any propane or propanol conjugatedmoiety can be included in Z or Z′.

In additional embodiments, each of Z and/or Z′ includes a combination ofan abasic moiety and an unmodified deoxyribonucleotide or ribonucleotideor a combination of a hydrocarbon moiety and an unmodifieddeoxyribonucleotide or ribonucleotide or a combination of an abasicmoiety (deoxyribo or ribo) and a hydrocarbon moiety. In suchembodiments, each of Z and/or Z′ includes C3-rAb or C3-dAb wherein eachmoiety is covalently bond to the adjacent moiety via a phospho-basedbond, preferably a phosphodiester, phosphorothioate or phosphonoacetatebond.

In certain embodiments, nucleic acid molecules as disclosed hereininclude a sense oligonucleotide sequence selected from any one ofoligonucleotide shown infra in Tables 4 and 5 (SEQ ID NOS:60-126 and194-218).

In certain preferred embodiments, compounds provided include Compound_1,Compound_2, Compound_3, Compound_4, Compound_5, Compound_6, Compound_7,Compound_8, and Compound_9, described infra.

In some embodiments, (such as, for example, Compound_1, Compound_5, andCompound_6) provided are 19-mer double-stranded nucleic acid moleculeswherein the antisense strand is SEQ ID NO:127 and the sense strand isSEQ ID NO:60. In certain embodiments, provided are 19-merdouble-stranded nucleic acid molecules wherein the antisense strand isSEQ ID NO:127 and includes 2′OMe modified ribonucleotides; a2′-5′-ribonucleotide in at least one of positions 1, 5, 6, or 7; and anon-nucleotide moiety covalently attached to the 3′-terminus; and thesense strand is SEQ ID NO:60 and includes at least one2′-5′-ribonucleotide or 2′OMe modified ribonucleotide; a non-nucleotidemoiety covalently attached at the 3′-terminus; and a cap moietycovalently attached at the 5′-terminus. In some embodiments, providedare 19-mer double-stranded nucleic acid molecule wherein the antisensestrand is SEQ ID NO:127; and includes 2′OMe modified ribonucleotides atpositions 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide inposition 7; and a C3OH non-nucleotide moiety covalently attached at the3′-terminus; and the sense strand is SEQ ID NO:60 and includes fiveconsecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16,17, 18, and 19; a C3Pi non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic moiety covalently attached at the5′-terminus.

In one embodiment, provided is Compound_1 that is a 19-merdouble-stranded nucleic acid molecule wherein the antisense strand isSEQ ID NO:127 and includes 2′OMe modified ribonucleotides at positions3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7;and a C3Pi-C3OH non-nucleotide moiety covalently attached at the3′-terminus; and the sense strand is SEQ ID NO:60 and includes fiveconsecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16,17, 18, and 19; a C3Pi non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic moiety covalently attached at the5′-terminus; and that further includes a 2′OMe modified ribonucleotideat position 1 of the antisense strand.

In one embodiment, provided is Compound_6 that is a 19-merdouble-stranded nucleic acid molecule wherein the antisense strand isSEQ ID NO:127 and includes 2′OMe modified ribonucleotides at positions3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7;and a C3Pi-C3OH non-nucleotide moiety covalently attached at the3′-terminus; and the sense strand is SEQ ID NO:60 and includes fiveconsecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16,17, 18, and 19; a C3Pi non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic moiety covalently attached at the5′-terminus; and that further includes a 2′-5′-ribonucleotide atposition 1 of the antisense strand.

In one embodiment, provided is Compound_5 that is a 19-merdouble-stranded nucleic acid molecule wherein the antisense strand isSEQ ID NO:127 and includes 2′OMe modified ribonucleotides in positions1, 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position7; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the3′-terminus; and the sense strand is SEQ ID NO:60 and includes 2′OMemodified ribonucleotides in positions 7, 13, 16 and 18; a2′-5′-ribonucleotide at position 9; a C3OH non-nucleotide moietycovalently attached at the 3′-terminus; and an inverted abasic moietycovalently attached at the 5′-terminus.

In some embodiments, (such as, for example, Compound_2, and Compound_7,described infra) provided are 19-mer double-stranded nucleic acidmolecules wherein the sense strand is SEQ ID NO:63 and the antisensestrand is SEQ ID NO:130. In some embodiments provided are 19-merdouble-stranded nucleic acid molecules wherein the sense strand is SEQID NO:63 and includes 2′OMe modified pyrimidine ribonucleotides; anon-nucleotide moiety covalently attached at the 3′-terminus; and a capmoiety covalently attached at the 5′-terminus; and the antisense strandis SEQ ID NO:130 and includes 2′OMe modified ribonucleotides; a2′-5′-ribonucleotide at position 7; and a non-nucleotide moietycovalently attached at the 3′-terminus. In some embodiments provided are19-mer double-stranded nucleic acid molecules wherein the sense strandis SEQ ID NO:63 and includes 2′OMe modified ribonucleotides; anon-nucleotide moiety covalently attached at the 3′-terminus; and a capmoiety covalently attached at the 5′-terminus; and the antisense strandis SEQ ID NO:130 and includes 2′OMe modified ribonucleotides; a2′-5′-ribonucleotide in at least one of positions 5, 6 or 7; and anon-nucleotide moiety covalently attached at the 3′-terminus.

In one embodiment, provided is Compound_2 that is a 19-merdouble-stranded nucleic acid molecule wherein the sense strand is SEQ IDNO:63 and includes 2′OMe modified ribonucleotides in positions 2, 14 and18; a C3OH moiety covalently attached at the 3′-terminus; and aninverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand is SEQ ID NO:130 and includes2′OMe modified ribonucleotides in positions 1, 3, 5, 9, 12, 13, and 17;a 2′-5′-ribonucleotide in at least one of positions 5, 6 or 7; andC3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus.

In one embodiment, provided is Compound_7 that is a 19-merdouble-stranded nucleic acid molecule wherein the sense strand is SEQ IDNO:63 and includes 2′OMe modified ribonucleotides in positions 2, 14 and18; a C3OH moiety covalently attached at the 3′-terminus; and aninverted abasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand is SEQ ID NO:130 and includes2′OMe modified ribonucleotides in positions 1, 3, 5, 9, 11, 13, and 17;a 2′-5′-ribonucleotide at position 7; and a C3Pi-C3OH non-nucleotidemoiety covalently attached at the 3′-terminus.

In some embodiments, (such as, for example, Compound_3, described infra)provided are 19-mer double-stranded nucleic acid molecules wherein thesense strand is SEQ ID NO:98 and the antisense strand is SEQ ID NO:165.In some embodiments, provided are 19-mer double-stranded nucleic acidmolecules wherein the sense strand is SEQ ID NO:98 and includes2′-5′-ribonucleotides in positions at the 3′-terminus; a non-nucleotidemoiety covalently attached at the 3′-terminus; and a cap moietycovalently attached at the 5′-terminus; and the antisense strand is SEQID NO:165 and includes 2′OMe modified ribonucleotides; a2′-5′-ribonucleotide in at least one of positions 5, 6 or 7; and anon-nucleotide moiety covalently attached at the 3′-terminus. In oneembodiment, provided is Compound_3 that is a 19-mer double-strandednucleic acid molecule wherein the sense strand is SEQ ID NO:98 andincludes 2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; aC3-OH 3′ moiety covalently attached at the 3′-terminus; and an invertedabasic deoxyribonucleotide moiety covalently attached at the5′-terminus; and the antisense strand is SEQ ID NO:165 and includes2′OMe modified ribonucleotides in positions 2, 4, 6, 8, 11, 13, 15, 17,and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH covalentlyattached at the 3′-terminus.

In some embodiments, (such as, for example, Compound_4, Compound_8 andCompound_9, described infra) provided are 19-mer double-stranded nucleicacid molecules wherein the sense strand is SEQ ID NO:101 and theantisense strand is SEQ ID NO:168. In some embodiments provided are19-mer double-stranded nucleic acid molecules wherein the sense strandis SEQ ID NO:101 and includes 2′OMe modified pyrimidine ribonucleotides;an optional 2′-5′-ribonucleotide in one of position 9 or 10; anon-nucleotide moiety covalently attached at the 3′-terminus; and a capmoiety covalently attached at the 5′-terminus; and the antisense strandis SEQ ID NO:168 and includes 2′OMe modified ribonucleotides; a2′-5′-ribonucleotide in at least one of positions 5, 6, or 7; and anon-nucleotide moiety covalently attached at the 3′-terminus.

In one embodiment, provided is Compound_4 that is a 19-merdouble-stranded nucleic acid molecule wherein sense strand is SEQ IDNO:101 and includes 2′OMe modified ribonucleotides in positions 4, 11,13, and 17; a 2′-5′-ribonucleotide in position 9; a C3OH non-nucleotidemoiety covalently attached at the 3′-terminus; and an inverted abasicdeoxyribonucleotide moiety covalently attached at the 5′-terminus; andthe antisense strand is SEQ ID NO:168 and includes 2′OMe modifiedribonucleotides in positions 1, 4, 8, 11 and 15; a 2′-5′-ribonucleotidein position 6; a C3Pi-C3OH overhang covalently attached at the3′-terminus.

In one embodiment, provided is Compound_8 that is a 19-merdouble-stranded nucleic acid molecule wherein sense strand is SEQ IDNO:101 and includes 2′OMe modified ribonucleotides in positions 4, 11,13, and 17; a C3OH non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand is SEQID NO:168 and includes 2′OMe modified ribonucleotides in positions 1, 4,8, 13 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OHoverhang covalently attached at the 3′-terminus.

In one embodiment, provided is Compound_9 that is a 19-merdouble-stranded nucleic acid molecule wherein the sense strand is SEQ IDNO:101 and includes 2′OMe modified ribonucleotides in positions 2, 4,11, 13, and 17; a C3OH non-nucleotide moiety covalently attached at the3′-terminus; and an inverted abasic deoxyribonucleotide moietycovalently attached at the 5′-terminus; and the antisense strand is SEQID NO:168 and includes 2′OMe modified ribonucleotides in positions 1, 4,8, 11 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OHmoiety covalently attached at the 3′-terminus.

In another aspect, provided are methods for reducing the expression ofhsp47 in a cell by introducing into a cell a nucleic acid molecule asprovided herein in an amount sufficient to reduce expression of hsp47.In one embodiment, the cell is hepatic stellate cell. In anotherembodiment, the cell is a stellate cell in renal or pulmonary tissue. Incertain embodiment, the method is performed in vitro. In anotherembodiment, the method is performed in vivo.

In another aspect, provided are methods for treating an individualsuffering from a disease associated with hsp47. The methods includeadministering to the individual a nucleic acid molecule such as providedherein in an amount sufficient to reduce expression of hsp47. In certainembodiments, the disease associated with hsp47 is a disease selectedfrom the group consisting of liver fibrosis, cirrhosis, pulmonaryfibrosis including lung fibrosis (including ILF), any condition causingkidney fibrosis (e.g., CKD including ESRD), peritoneal fibrosis, chronichepatic damage, fibrillogenesis; fibrotic diseases in other organs;abnormal scarring (keloids) associated with all possible types of skininjury, accidental and iatrogenic (operations), scleroderma,cardiofibrosis, failure of glaucoma filtering operation, and intestinaladhesions. In some embodiments, the compounds may be useful in treatingorgan-specific indications, for example, indications including thoseshown in Table 1 below, listing organs and respective indications:

TABLE 1 Skin Pathologic scarring as keloid and hypertrophic scarSurgical scarring Injury scarring keloid, or nephrogenic fibrosingdermatopathy Peritoneum Peritoneal fibrosis Adhesions PeritonealSclerosis associated with continual ambulatory peritoneal dialysis(CAPD) Liver Cirrhosis including post-hepatitis C cirrhosis, primarybiliary cirrhosis Liver fibrosis, e.g. Prevention of Liver Fibrosis inHepatitis C carriers schistomasomiasis cholangitis Liver cirrhosis dueto Hepatitis C post liver transplant or Non-Alcoholic Steatohepatitis(NASH) Pancreas inter(peri)lobular fibrosis (e.g., alcoholic chronicpancreatitis), periductal fibrosis (e.g., hereditary pancreatitis),periductal and interlobular fibrosis (e.g., autoimmune pancreatitis),diffuse inter- and intralobular fibrosis (e.g., obstructive chronicpancreatitis) Kidney Chronic Kidney Disease (CKD) of any etiology.Treatment of early stage CKD (elevated SCr) in diabetic patients(prevent further deterioration in renal function) kidney fibrosisassociated with lupus glomeruloschelerosis Diabetic Nephropathy HeartCongestive heart failure, Endomyocardial fibrosis, cardiofibrosisfibrosis associated with myocardial infarction Lung Asthma, Idiopathicpulmonary fibrosis (IPF); Interstitial lung fibrosis (ILF) RadiationPneumonitis leading to Pulmonary Fibrosis (e.g. due to cancer treatingradiation) Bone marrow Myeloproliferative disorders: Myelofibrosis (MF),Polycythemia vera (PV), Essential thrombocythemia (ET) idiopathicmyelofibrosis drug induced myelofibrosis. Eye Anterior segment: Cornealopacification e,g, following inherited dystrophies, herpetic keratitisor ptery gia; Glaucoma Posterior segment fibrosis and traction retinaldetachment, a complication of advanced diabetic retinopathy (DR);Fibrovascular scarring and gliosis in the retina; Under the retinafibrosis for example subsequent to subretinal hemorrhage associated withneovascular AMD Retro-orbital fibrosis, postcataract surgery,proliferative vitreoretinopathy. Ocular cicatricial pemphigoid IntestineIntestinal fibrosis, Crohn's disease Vocal cord Vocal cord scarring,vocal cord mucosal fibrosis, laryngeal fibrosis VasculatureAtherosclerosis, postangioplasty arterial restenosis Multi systemicScleroderma systemic sclerosis; multifocal fibrosclerosis;sclerodermatous graft-versus-host disease in bone marrow transplantrecipients, and nephrogenic systemic fibrosis (exposure togadolinium-based contrast agents (GBCAs), 30% of MRIs) Malignancies ofvarious origin Metastatic and invasive cancer by inhibiting function ofactivated tumor associated myofibroblasts

Another embodiment of the description is a method for treating astellate cell-related disorder, the method comprising administering aneffective amount of the pharmaceutical composition described infra to asubject in need thereof. The disorder includes hepatitis, hepaticfibrosis, hepatic cirrhosis, liver cancer, pancreatitis, pancreaticfibrosis, pancreatic cancer, vocal cord scarring, vocal cord mucosalfibrosis, and laryngeal fibrosis. In some embodiments the preferredindications include, liver cirrhosis due to Hepatitis C post livertransplant; liver cirrhosis due to Non-Alcoholic Steatohepatitis (NASH);idiopathic pulmonary fibrosis; radiation pneumonitis leading topulmonary fibrosis; diabetic nephropathy; peritoneal sclerosisassociated with continual ambulatory peritoneal dialysis (CAPD) andocular cicatricial pemphigoid.

Fibrotic liver indications include Alcoholic Cirrhosis, Hepatitis Bcirrhosis, Hepatitis C cirrhosis, Hepatitis C (Hep C) cirrhosispost-orthotopic liver transplant (OLTX), non-alcoholicsteatohepatitis/nonalcoholic fatty liver disease (NASH/NAFLD), primarybiliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliaryatresia, alpha-1 antitrypsin deficiency (A1AD), copper storage diseases(Wilson's disease), fructosemia, galactosemia, glycogen storage diseases(especially types III, IV, VI, IX, and X), iron-overload syndromes(hemochromatosis), lipid abnormalities (e.g., Gaucher's disease),peroxisomal disorders (e.g., Zellweger syndrome), tyrosinemia,congenital hepatic fibrosis, bacterial Infections (e.g., brucellosis),parasitic (e.g., echinococcosis), Budd-Chiari syndrome (hepaticveno-occlusive disease).

Pulmonary indications include Idiopathic Pulmonary Fibrosis, Silicosis,Pneumoconiosis, bronchopulmonary dysplasia in newborn following neonatalrespiratory distress syndrome, bleomycin/chemolung injury, bronchiolitisobliterans (BOS) Post-lung transplant, Chronic obstructive pulmonarydisorder (COPD), cystic fibrosis, and asthma.

Cardiac indications include cardiomyopathy, atherosclerosis (Bergersdisease, etc), endomyocardial fibrosis, atrial fibrillation, scarringpost-myocardial infarction (MI).

Other Thoracic indications include Radiation-induced capsule tissuereactions around textured breast implants, and oral submucosal fibrosis.

Renal indications include autosomal dominant polycystic kidney disease(ADPKD), Diabetic nephropathy (diabetic glomerulosclerosis), focalsegmental glomerulosclerosis (FSGS) (collapsing vs. other histologicvariants), IgA Nephropathy (Berger Disease), Lupus Nephritis, Wegener's,Scleroderma, Goodpasture Syndrome, tubulointerstitial fibrosis: druginduced (protective) pencillins, cephalosporins, analgesic nephropathy,membrano-proliferative glomerulonephritis (MPGN), Henoch-Schonleinpurpura, Congenital nephropathies: Medullary Cystic Disease,Nail-Patella Syndrome and Alport Syndrome.

Bone Marrow indications include lymphangioleiomyositosis (LAM), chronicgraft vs. host disease, polycythemia vera, essential thrombocythemia,and myelofibrosis.

Ocular indications include retinopathy of prematurity (RoP), ocularcicatricial pemphigoid, Lacrimal gland fibrosis, Retinal attachmentsurgery, corneal opacity, herpetic keratitis, pterygia, Glaucoma,age-related macular degeneration (AMD/ARMD), retinal fibrosis associateddiabetes mellitus (DM) retinopathy.

Gynecological indications include Endometriosis add on to hormonaltherapy for prevention of scarring, post-STD fibrosis/salpingitis.

Systemic indications include Dupuytren's disease, Palmar fibromatosis,Peyronie's disease, Ledderhose disease, keloids, multifocalfibrosclerosis, nephrogenic systemic fibrosis and myelofibrosis(anemia).

Injury-associated fibrotic diseases include burn-induced (chemicalincluded) skin and soft tissue scarring and contraction,radiation-induced skin and organ scarring, post cancer therapeuticradiation treatment, keloid (skin).

Surgical indications include peritoneal fibrosis post-peritonealdialysis catheter, corneal implant, cochlear implant, other implants,silicone implants in breasts, chronic sinusitis; adhesions,pseudointimal hyperplasia of dialysis grafts.

Other indications include chronic pancreatitis.

In some embodiments, provided is a method for treatment of a subjectsuffering from liver fibrosis comprising administering to the subject aneffective amount of a nucleic acid molecule disclosed herein, therebytreating liver fibrosis. In some embodiments, the subject is sufferingfrom cirrhosis of the liver due to hepatitis. In some embodiments, thesubject is suffering from cirrhosis of the liver due to NASH.

In some embodiments, provided is the use of a nucleic acid moleculedisclosed herein for the manufacture of a medicament to treat liverfibrosis. In some embodiments, the liver fibrosis is due to hepatitis.In some embodiments, the liver fibrosis is due to NASH.

In some embodiments, provided is a method for remodeling of scar tissuecomprising administering to a subject in need thereof an effectiveamount of a nucleic acid molecule disclosed herein, thereby effectingscar tissue remodeling. In some embodiments, the scar tissue is in theliver. In some embodiments, the subject is suffering from cirrhosis ofthe liver due to hepatitis. In some embodiments, the subject issuffering from cirrhosis of the liver due to NASH.

In some embodiments, a method for modulating fibrosis regression isprovided comprising administering to a subject in need thereof aneffective amount of a nucleic acid molecule disclosed herein, therebyeffecting fibrosis regression.

In some embodiments, provided is a method for reduction of scar tissuein a subject comprising the step of administering to the subject aneffective amount of a nucleic acid molecule disclosed herein to reducethe scar tissue. In some embodiments, provided is a method for reducingscar tissue in a subject comprising the step of topically applying toscar tissue an effective amount of a nucleic acid molecule disclosedherein to reduce scar tissue.

In some embodiments, provided is a method for improving the appearanceof scar tissue comprising the step of topically applying to scar tissuean effective amount of a nucleic acid molecule disclosed herein toimprove the appearance of the scar tissue.

In some embodiments, provided is a method for treatment of a subjectsuffering from lung fibrosis comprising administering to the subject aneffective amount of a nucleic acid molecule disclosed herein, therebytreating the lung fibrosis. In some embodiments, the subject issuffering from interstitial lung fibrosis (ILF). In some embodiments,the subject is suffering from Radiation Pneumonitis leading to PulmonaryFibrosis. In some embodiments, the subject is suffering from druginduced lung fibrosis.

In some embodiments, provided is the use of a nucleic acid moleculedisclosed herein for the manufacture of a medicament to treat lungfibrosis. In some embodiments, the lung fibrosis is ILF. In someembodiments, the lung fibrosis is drug- or radiation-induced lungfibrosis.

In one aspect, provided are pharmaceutical compositions that include anucleic acid molecule (e.g., a siNA molecule) as described herein in apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical formulation includes, or involves, a delivery systemsuitable for delivering nucleic acid molecules (e.g., siNA molecules) toan individual such as a patient; for example delivery systems describedin more detail below.

In a related aspect, provided are compositions or kits that include anucleic acid molecule (e.g., an siNA molecule) packaged for use by apatient. The package may be labeled or include a package label or insertthat indicates the content of the package and provides certaininformation regarding how the nucleic acid molecule (e.g., an siNAmolecule) should be or can be used by a patient, for example the labelmay include dosing information and/or indications for use. In certainembodiments, the contents of the label will bear a notice in a formprescribed by a government agency, for example the United States Foodand Drug administration (FDA). In certain embodiments, the label mayindicate that the nucleic acid molecule (e.g., an siNA molecule) issuitable for use in treating a patient suffering from a diseaseassociated with hsp47; for example, the label may indicate that thenucleic acid molecule (e.g., an siNA molecule) is suitable for use intreating fibroids; or for example the label may indicate that thenucleic acid molecule (e.g., an siNA molecule) is suitable for use intreating a disease selected from the group consisting of fibrosis, liverfibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritonealfibrosis, chronic hepatic damage, and fibrillogenesis.

As used herein, the term “heat shock protein 47” or “hsp47” or “HSP47”are used interchangeably and refer to any heat shock protein 47,peptide, or polypeptide having any hsp47 protein activity. Heat shockprotein 47 is a serine proteinase inhibitor (serpin) also known, forexample, as serpin peptidase inhibitor, clade H, member 1 (SERPINH1),SERPINH2, collagen binding protein 1 (CBP1), CBP2, gp46;arsenic-transactivated protein 3 (AsTP3); HSP47; proliferation-inducinggene 14 (PIG14); PPROM; rheumatoid arthritis antigen A-47 (RA-A47);colligin-1; and colligin-2. In certain preferred embodiments, “hsp47”refers to human hsp47. Heat shock protein 47 (or more particularly humanhsp47) may have an amino acid sequence that is the same, orsubstantially the same, as SEQ ID NO. 2.

As used herein the term “nucleotide sequence encoding hsp47” means anucleotide sequence that codes for an hsp47 protein, or portion thereof.The term “nucleotide sequence encoding hsp47” is also meant to includehsp47 coding sequences such as hsp47 isoforms, mutant hsp47 genes,splice variants of hsp47 genes, and hsp47 gene polymorphisms. A nucleicacid sequence encoding hsp47 includes mRNA sequences encoding hsp47,which can also be referred to as “hsp47 mRNA.” An exemplary sequence ofhuman hsp47 mRNA is SEQ ID. NO. 1.

As used herein, the term “nucleic acid molecule” or “nucleic acid” areused interchangeably and refer to an oligonucleotide, nucleotide orpolynucleotide. Variations of “nucleic acid molecule” are described inmore detail herein. A nucleic acid molecule encompasses both modifiednucleic acid molecules and unmodified nucleic acid molecules asdescribed herein. A nucleic acid molecule may includedeoxyribonucleotides, ribonucleotides, modified nucleotides ornucleotide analogs in any combination.

As used herein, the term “nucleotide” refers to a chemical moiety havinga sugar (or an analog thereof, or a modified sugar), a nucleotide base(or an analog thereof, or a modified base), and a phosphate group (oranalog thereof, or a modified phosphate group). A nucleotide encompassesmodified nucleotides or unmodified nucleotides as described herein. Asused herein, nucleotides may include deoxyribonucleotides (e.g.,unmodified deoxyribonucleotides), ribonucleotides (e.g., unmodifiedribonucleotides), and modified nucleotide analogs including, inter alia,LNA and UNA, peptide nucleic acids, L-nucleotides (also referred to asmirror nucleotides), ethylene-bridged nucleic acid (ENA), arabinoside,PACE, nucleotides with a 6 carbon sugar, as well as nucleotide analogs(including abasic nucleotides) often considered to be non-nucleotides.In some embodiments, nucleotides may be modified in the sugar,nucleotide base and/or in the phosphate group with any modificationknown in the art, such as modifications described herein. A“polynucleotide” or “oligonucleotide” as used herein, refers to a chainof linked nucleotides; polynucleotides and oligonucleotides may likewisehave modifications in the nucleotide sugar, nucleotide bases andphosphate backbones as are well known in the art and/or are disclosedherein.

As used herein, the term “short interfering nucleic acid”, “siNA”, or“short interfering nucleic acid molecule” refers to any nucleic acidmolecule capable of modulating gene expression or viral replication.Preferably siNA inhibits or down regulates gene expression or viralreplication. siNA includes without limitation nucleic acid moleculesthat are capable of mediating sequence specific RNAi, for example shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),short hairpin RNA (shRNA), short interfering oligonucleotide, shortinterfering nucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. As used herein, “short interfering nucleic acid”,“siNA”, or “short interfering nucleic acid molecule” has the meaningdescribed in more detail elsewhere herein.

As used herein, the term “complementary” means that a nucleic acid canform hydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or other non-traditional types. In reference tothe nucleic acid molecules disclosed herein, the binding free energy fora nucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Fully complementary”means that all the contiguous residues of a nucleic acid sequence willhydrogen bond with the same number of contiguous residues in a secondnucleic acid sequence. In one embodiment, a nucleic acid moleculedisclosed herein includes about 15 to about 35 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 or more) nucleotides that are complementary to one or moretarget nucleic acid molecules or a portion thereof

As used herein, the term “sense region” refers to a nucleotide sequenceof a siNA molecule complementary (partially or fully) to an antisenseregion of the siNA molecule. The sense strand of a siNA molecule caninclude a nucleic acid sequence having homology with a target nucleicacid sequence. As used herein, “sense strand” refers to nucleic acidmolecule that includes a sense region and may also include additionalnucleotides. Nucleotide positions of the sense strand are hereinnumbered 5′>3′.

As used herein, the term “antisense region” refers to a nucleotidesequence of a siNA molecule complementary (partially or fully) to atarget nucleic acid sequence. The antisense strand of a siNA moleculecan optionally include a nucleic acid sequence complementary to a senseregion of the siNA molecule. As used herein, “antisense strand” refersto nucleic acid molecule that includes an antisense region and may alsoinclude additional nucleotides. Nucleotide positions of the antisensestrand are herein numbered 5′>3′.

As used herein, the term “RNA” refers to a molecule that includes atleast one ribonucleotide residue.

As used herein, the term “duplex region” refers to the region in twocomplementary or substantially complementary oligonucleotides that formbase pairs with one another, either by Watson-Crick base pairing or anyother manner that allows for a duplex between oligonucleotide strandsthat are complementary or substantially complementary. For example, anoligonucleotide strand having 21 nucleotide units can base pair withanother oligonucleotide of 21 nucleotide units, yet only 19 bases oneach strand are complementary or substantially complementary, such thatthe “duplex region” consists of 19 base pairs. The remaining base pairsmay, for example, exist as 5′ and 3′ overhangs. Further, within theduplex region, 100% complementarity is not required; substantialcomplementarity is allowable within a duplex region. Substantialcomplementarity refers to complementarity between the strands such thatthey are capable of annealing under biological conditions. Techniques toempirically determine if two strands are capable of annealing underbiological conditions are well known in the art. Alternatively, twostrands can be synthesized and added together under biologicalconditions to determine if they anneal to one another.

As used herein, the terms “non-pairing nucleotide analog” means anucleotide analog which includes a non-base pairing moiety including butnot limited to: 6-des-amino adenosine (nebularine), 4-Me-indole,3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC. In someembodiments the non-base pairing nucleotide analog is a ribonucleotide.In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes withoutlimitation a halogen, alcohol, amine, carboxylic, ester, amide,aldehyde, ketone, ether groups.

An “abasic nucleotide” or “abasic nucleotide analog” is as used hereinmay also be often referred to herein and in the art as apseudo-nucleotide or an unconventional moiety. While a nucleotide is amonomeric unit of nucleic acid, generally consisting of a ribose ordeoxyribose sugar, a phosphate, and a base (adenine, guanine, thymine,or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), anabasic or pseudo-nucleotide lacks a base, and thus is not strictly anucleotide as the term is generally used in the art. Abasic deoxyribosemoieties include for example, abasic deoxyribose-3′-phosphate;1,2-dideoxy-D-ribofuranose-3-phosphate;1,4-anhydro-2-deoxy-D-ribitol-3-phosphate. Inverted abasic deoxyribosemoieties include inverted deoxyriboabasic; 3′,5′ inverted deoxyabasic5′-phosphate.

The term “capping moiety” (z″) as used herein includes a moiety whichcan be covalently linked to the 5′-terminus of (N′)_(y) and includesabasic ribose moiety, abasic deoxyribose moiety, modifications abasicribose and abasic deoxyribose moieties including 2′ O alkylmodifications; inverted abasic ribose and abasic deoxyribose moietiesand modifications thereof; C₆-imino-Pi; a mirror nucleotide includingL-DNA and L-RNA; 5′OMe nucleotide; and nucleotide analogs including4′,5′-methylene nucleotide; 1-(β-D-erythrofuranosyl)nucleotide; 4′-thionucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate;1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexylphosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate;1,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosylnucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutylnucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted abasicmoiety; 1,4-butanediol phosphate; 5′-amino; and bridging or non-bridgingmethylphosphonate and 5′-mercapto moieties.

Certain capping moieties may be abasic ribose or abasic deoxyribosemoieties; inverted abasic ribose or abasic deoxyribose moieties;C6-amino-Pi; a mirror nucleotide including L-DNA and L-RNA. The nucleicacid molecules as disclosed herein may be synthesized using one or moreinverted nucleotides, for example inverted thymidine or inverted adenine(for example see Takei, et al., 2002. JBC 277(26):23800-06).

The term “unconventional moiety” as used herein refers to non-nucleotidemoieties including an abasic moiety, an inverted abasic moiety, ahydrocarbon (alkyl) moiety and derivatives thereof, and further includesa deoxyribonucleotide, a modified deoxyribonucleotide, a mirrornucleotide (L-DNA or L-RNA), a non-base pairing nucleotide analog and anucleotide joined to an adjacent nucleotide by a 2′-5′ internucleotidephosphate bond; bridged nucleic acids including LNA and ethylene bridgednucleic acids, linkage modified (e.g. PACE) and base modifiednucleotides as well as additional moieties explicitly disclosed hereinas unconventional moieties.

As used herein, the term “inhibit”, “down-regulate”, or “reduce” withrespect to gene expression means the expression of the gene, or level ofRNA molecules or equivalent RNA molecules encoding one or more proteinsor protein subunits (e.g., mRNA), or activity of one or more proteins orprotein subunits, is reduced below that observed in the absence of aninhibitory factor (such as a nucleic acid molecule, e.g., an siNA, forexample having structural features as described herein); for example theexpression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,10%, 5% or less than that observed in the absence of an inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a protocol with respect to assessment of theeffect of gp46-siRNA in vitro using NRK cells, and determination ofoptimal sequence, timing, and concentration.

FIG. 2 is a photographic diagram showing the result of western blottingof gp46 and actin (24 hour culturing, examination of optimal sequence).

FIG. 3 is a photographic diagram showing the result of western blottingof gp46 and actin (24 hour culturing, examination of optimalconcentration).

FIG. 4 is a photographic diagram showing the result of western blottingof gp46 and actin (concentration 50 nM, examination of optimal culturingtime).

FIG. 5 is a diagram showing a protocol for evaluating inhibition ofexpression of collagen by gp46-siRNA in NRK cells.

FIG. 6 is a graph showing inhibition of collagen synthesis by siRNA.

FIG. 7 is a photographic diagram showing HSC-specific siRNAtransfection.

FIG. 8 is a photographic diagram for evaluating HSC-specific siRNAtransfection percentage.

FIG. 9 is a photographic diagram for evaluating inhibition of expressionof gp46 by siRNA.

FIG. 10 is a photographic diagram showing azan staining of rat liver towhich DMN had been administered.

FIG. 11 is a diagram showing an LC rat treatment protocol.

FIG. 12 is a photographic diagram showing azan staining of LC rat liverto which VA-Lip-gp46siRNA had been administered.

FIG. 13. is a diagram showing a method for extracting a stained portionby means of NIH Image (6 positions being randomly taken from anazan-stained image).

FIG. 14 is a graph showing the ratio by area occupied by fibroticportions in liver histology (Collagen ratio by area, %).

FIG. 15 is a graph showing the amount of hydroxyproline in hepatictissue.

FIG. 16 is a graph showing a survival curve for hepatic cirrhosis rat towhich VA-Lip-gp46siRNA had been intraportally administered.

FIG. 17 is a photographic diagram showing azan staining of hepatictissue of hepatic cirrhosis rat to which VA-Lip-gp46siRNA had beenintraportally administered.

FIG. 18 is a graph showing a survival curve for hepatic cirrhosis rat towhich VA-Lip-gp46siRNA had been intraportally administered.

FIG. 19 is a photographic diagram showing azan staining of hepatictissue of hepatic cirrhosis rat to which VA-Lip-gp46siRNA had beenintraportally administered.

FIG. 20 is a graph showing a survival curve for hepatic cirrhosis rat towhich VA-Lip-gp46siRNA had been intravenously administered.

FIG. 21 is a graph showing a survival curve for hepatic cirrhosis rat towhich VA-Lip-gp46siRNA had been intravenously administered.

FIG. 22 is a photographic diagram showing azan staining of hepatictissue of hepatic cirrhosis rat to which VA-Lip-gp46siRNA had beenintravenously administered.

FIG. 23 is a diagram showing improvement of VA-Lip-gp46siRNAtransfection efficiency by RBP.

FIG. 24 is a diagram showing inhibition of VA-Lip-gp46siRNA transfectionby anti-RBP antibody.

FIG. 25 is a bar graph showing the effect of GFP siNA on variousreporter cell lines. Cell lines were established by lenti-viralinduction of human HSP47 cDNA-GFP or rat GP46 cDNA-GFP construct intoHEK293, human fibrosarcoma cell line HT1080, human HSC line hTERT or NRKcell line. Negative control siNA or siNA against GFP was introduced intothe cells and GFP fluorescence was measured. The results showed thatsiNA against GFP knocks down the fluorescence to different extent indifferent cell lines. 293 HSP47-GFP and 293_GP46-GFP cell lines wereselected for siHsp47 screening due to their easiness of beingtransfected and sensitivity to fluorescence knockdown.

FIGS. 26A, 26B, 26C, and 26D are a series of bar graphs showing thecytotoxicity and knockdown efficiency of various siHsp47s in 293HSP47-GFP and 293_GP46-GFP cell lines. The result showed that siHsp47-C,siHsp47-2 and siHsp47-2d efficiently knockdown both human HSP47 and ratGP46 (the human hsp47 homolog) without substantial cytotoxicity. siGp46Aagainst GP46 does not knock down human HSP47. Additionally, the newlydesigned siHsp47s outperformed siGp46A in knocking down rat GP46.

FIG. 27 is a bar graph showing the knock down effect of various siHsp47son hsp47 mRNA, measured by TAQMAN® qPCR using the human HSC cell linehTERT. The Y axis represents the remaining mRNA level of hsp47. HSP47-Cwas most effective among all the hsp47 siNAs tested.

FIG. 28 is a bar graph showing the effect of different hsp47 siNAs oncollagen I expression in hTERT cells. The level of collagen I mRNAlevels were measured by real-time quantitative PCR using TAQMAN® probe.The Y axis represents the remaining mRNA expression level of collagen I.The result showed that collagen I mRNA level is significantly reduced inthe cells treated with some of the candidates (siHsp47-2, siHsp47-2d,and their combination with siHsp47-1).

FIG. 29 is a graph showing a decrease in fibrotic areas of the liver inanimals treated with siHSP47.

FIG. 30 is a schematic of the treatment schedule and evaluation methodused in Example 22.

FIG. 31 shows the results of histological staining with Azan in thepulmonary field. The pictures display representative lung field ofAzan-stained sections of each group at 80× magnification. (a)Pretreatment (BLM IT−2W); (b) Disease rat (BLM IT−5W+PBS i.v.); and (c)Treatment (BLM IT+siRNA i.v.), (d) Sham (Saline-IT+PBS i.v.).

FIG. 32 shows fibrosis scoring, showing the results of evaluating twentyrandomly selected lung fields under 80× magnification for each rat. Thebar graph summarizes the fibrosis scoring of Azan-stained section foreach group. Statistical analysis were used One-way-ANOVA Bonferronimulti comparison test using Prism5 software.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

RNA Interference and siNA Nucleic Acid Molecules

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The presence ofdsRNA in cells triggers the RNAi response through a mechanism that hasyet to be fully characterized.

Dicer is involved in the processing of the dsRNA into short pieces ofdsRNA known as short interfering RNAs (siRNAs) (Zamore et al., 2000,Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein et al., 2001,Nature, 409, 363). siRNAs derived from dicer activity can be about 21 toabout 23 nucleotides in length and include about 19 base pair duplexes(Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, GenesDev., 15, 188). Dicer has also been implicated in the excision of 21-and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex, commonly referred to as an RNA-inducedsilencing complex (RISC), which mediates cleavage of single-stranded RNAhaving sequence complementary to the antisense strand of the siRNAduplex. Cleavage of the target RNA takes place in the middle of theregion complementary to the antisense strand of the siRNA duplex(Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Elbashir et al., 2001, Nature,411, 494 and Tuschl et al., WO0175164, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,WO0175164) has revealed certain requirements for siRNA length,structure, chemical composition, and sequence that are essential tomediate efficient RNAi activity.

Nucleic acid molecules (for example comprising structural features asdisclosed herein) may inhibit or down regulate gene expression or viralreplication by mediating RNA interference “RNAi” or gene silencing in asequence-specific manner. (See, e.g., Zamore et al., 2000, Cell, 101,25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature,411, 494-498; and Kreutzer et al., WO0044895; Zernicka-Goetz et al.,WO0136646; Fire, WO9932619; Plaetinck et al., WO0001846; Mello and Fire,WO0129058; Deschamps-Depaillette, WO9907409; and Li et al., WO0044914;Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science,297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al.,2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science,297, 1831).

An siNA nucleic acid molecule can be assembled from two separatepolynucleotide strands, where one strand is the sense strand and theother is the antisense strand in which the antisense and sense strandsare self-complementary (i.e., each strand includes nucleotide sequencethat is complementary to nucleotide sequence in the other strand); suchas where the antisense strand and sense strand form a duplex ordouble-stranded structure having any length and structure as describedherein for nucleic acid molecules as provided, for example wherein thedouble-stranded region (duplex region) is about 15 to about 49 (e.g.,about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49base pairs); the antisense strand includes nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid molecule(i.e., hsp47 mRNA) or a portion thereof and the sense strand includesnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof (e.g., about 17 to about 49 or more nucleotides of thenucleic acid molecules herein are complementary to the target nucleicacid or a portion thereof).

In certain aspects and embodiments, a nucleic acid molecule (e.g., asiNA molecule) provided herein may be a “RISC length” molecule or may bea Dicer substrate as described in more detail below.

An siNA nucleic acid molecule may include separate sense and antisensesequences or regions, where the sense and antisense regions arecovalently linked by nucleotide or non-nucleotide linkers molecules asis known in the art, or are alternately non-covalently linked by ionicinteractions, hydrogen bonding, van der Waals interactions, hydrophobicinteractions, and/or stacking interactions. Nucleic acid molecules mayinclude a nucleotide sequence that is complementary to nucleotidesequence of a target gene. Nucleic acid molecules may interact withnucleotide sequence of a target gene in a manner that causes inhibitionof expression of the target gene.

Alternatively, an siNA nucleic acid molecule is assembled from a singlepolynucleotide, where the self-complementary sense and antisense regionsof the nucleic acid molecules are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s), i.e., the antisense strandand the sense strand are part of one single polynucleotide that havingan antisense region and sense region that fold to form a duplex region(for example to form a “hairpin” structure as is well known in the art).Such siNA nucleic acid molecules can be a polynucleotide with a duplex,asymmetric duplex, hairpin or asymmetric hairpin secondary structure,having self-complementary sense and antisense regions, wherein theantisense region includes nucleotide sequence that is complementary tonucleotide sequence in a separate target nucleic acid molecule or aportion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence (e.g., a sequence ofhsp47 mRNA). Such siNA nucleic acid molecules can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region includes nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active nucleic acid molecule capable of mediating RNAi.

The following nomenclature is often used in the art to describe lengthsand overhangs of siNA molecules and may be used throughout thespecification and Examples. In all descriptions of oligonucleotidesherein, the identification of nucleotides in a sequence is given in the5′ to 3′ direction for both sense and antisense strands. Names given toduplexes indicate the length of the oligomers and the presence orabsence of overhangs. For example, a “21+2” duplex contains two nucleicacid strands both of which are 21 nucleotides in length, also termed a21-mer siRNA duplex or a 21-mer nucleic acid and having a 2 nucleotides3′-overhang. A “21-2” design refers to a 21-mer nucleic acid duplex witha 2 nucleotides 5′-overhang. A 21-0 design is a 21-mer nucleic acidduplex with no overhangs (blunt). A “21+2UU” is a 21-mer duplex with2-nucleotides 3′-overhang and the terminal 2 nucleotides at the 3′-endsare both U residues (which may result in mismatch with target sequence).The aforementioned nomenclature can be applied to siNA molecules ofvarious lengths of strands, duplexes and overhangs (such as 19-0, 21+2,27+2, and the like). In an alternative but similar nomenclature, a“25/27” is an asymmetric duplex having a 25 base sense strand and a 27base antisense strand with a 2-nucleotides 3′-overhang. A “27/25” is anasymmetric duplex having a 27 base sense strand and a 25 base antisensestrand.

Chemical Modifications

In certain aspects and embodiments, nucleic acid molecules (e.g., siNAmolecules) as provided herein include one or more modifications (orchemical modifications). In certain embodiments, such modificationsinclude any changes to a nucleic acid molecule or polynucleotide thatwould make the molecule different than a standard ribonucleotide or RNAmolecule (i.e., that includes standard adenosine, cytosine, uracil, orguanosine moieties); which may be referred to as an “unmodified”ribonucleotide or unmodified ribonucleic acid. Traditional DNA bases andpolynucleotides having a 2′-deoxy sugar represented by adenosine,cytosine, thymine, or guanosine moieties may be referred to as an“unmodified deoxyribonucleotide” or “unmodified deoxyribonucleic acid”;accordingly, the term “unmodified nucleotide” or “unmodified nucleicacid” as used herein refers to an “unmodified ribonucleotide” or“unmodified ribonucleic acid” unless there is a clear indication to thecontrary. Such modifications can be in the nucleotide sugar, nucleotidebase, nucleotide phosphate group and/or the phosphate backbone of apolynucleotide.

In certain embodiments, modifications as disclosed herein may be used toincrease RNAi activity of a molecule and/or to increase the in vivostability of the molecules, particularly the stability in serum, and/orto increase bioavailability of the molecules. Non-limiting examples ofmodifications include internucleotide or internucleoside linkages;deoxynucleotides or dideoxyribonucleotides at any position and strand ofthe nucleic acid molecule; nucleic acid (e.g., ribonucleic acid) with amodification at the 2′-position preferably selected from an amino,fluoro, methoxy, alkoxy and alkyl; 2′-deoxyribonucleotides, 2′OMeribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, biotingroup, and terminal glyceryl and/or inverted deoxy abasic residueincorporation, sterically hindered molecules, such as fluorescentmolecules and the like. Other nucleotides modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). Further details on variousmodifications are described in more detail below.

Modified nucleotides include those having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Sanger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). Non-limitingexamples of nucleotides having a northern configuration include LNAnucleotides (e.g., 2′-0, 4′-C-methylene-(D-ribofuranosyl) nucleotides);2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′OMe nucleotides. LNAs are described, for example, inElman et al., 2005; Kurreck et al., 2002; Crinelli et al., 2002; Braaschand Corey, 2001; Bondensgaard et al., 2000; Wahlestedt et al., 2000; andWO0047599, WO9914226, WO9839352, and WO04083430. In one embodiment, anLNA is incorporated at the 5′-terminus of the sense strand.

Chemical modifications also include UNAs, which are non-nucleotide,acyclic analogues, in which the C2′-C3′ bond is not present (althoughUNAs are not truly nucleotides, they are expressly included in the scopeof “modified” nucleotides or modified nucleic acids as contemplatedherein). In particular embodiments, nucleic acid molecules with anoverhang may be modified to have UNAs at the overhang positions (i.e., 2nucleotide overhang). In other embodiments, UNAs are included at the 3′-or 5′-ends. A UNA may be located anywhere along a nucleic acid strand,i.e. in position 7. Nucleic acid molecules may contain one or more UNA.Exemplary UNAs are disclosed in Nucleic Acids Symposium Series No. 52 p.133-134 (2008). In certain embodiments, nucleic acid molecules (e.g.,siNA molecules) as described herein, include one or more UNAs; or oneUNA. In some embodiments, a nucleic acid molecule (e.g., a siNAmolecule) as described herein that has a 3′-overhang include one or twoUNAs in the 3′ overhang. In some embodiments, a nucleic acid molecule(e.g., a siNA molecule) as described herein includes a UNA (for exampleone UNA) in the antisense strand; for example in position 6 or position7 of the antisense strand. Chemical modifications also includenon-pairing nucleotide analogs, for example as disclosed herein.Chemical modifications further include unconventional moieties asdisclosed herein.

Chemical modifications also include terminal modifications on the 5′and/or 3′ part of the oligonucleotides and are also known as cappingmoieties. Such terminal modifications are selected from a nucleotide, amodified nucleotide, a lipid, a peptide, and a sugar.

Chemical modifications also include six membered “six membered ringnucleotide analogs.” Examples of six-membered ring nucleotide analogsare disclosed in Allart, et al (Nucleosides & Nucleotides, 1998,17:1523-1526,; and Perez-Perez, et al., 1996, Bioorg. and Medicinal ChemLetters 6:1457-1460). Oligonucleotides including 6-membered ringnucleotide analogs including hexitol and altritol nucleotide monomersare disclosed in WO2006047842.

Chemical modifications also include “mirror” nucleotides which have areversed chirality as compared to normal naturally occurring nucleotide;that is, a mirror nucleotide may be an “L-nucleotide” analogue ofnaturally occurring D-nucleotide (see U.S. Pat. No. 6,602,858). Mirrornucleotides may further include at least one sugar or base modificationand/or a backbone modification, for example, as described herein, suchas a phosphorothioate or phosphonate moiety. U.S. Pat. No. 6,602,858discloses nucleic acid catalysts including at least one L-nucleotidesubstitution. Mirror nucleotides include for example L-DNA(L-deoxyriboadenosine-3′-phosphate (mirror dA);L-deoxyribocytidine-3′-phosphate (mirror dC);L-deoxyriboguanosine-3′-phosphate (mirror dG);L-deoxyribothymidine-3′-phosphate (mirror image dT)) and L-RNA(L-riboadenosine-3′-phosphate (mirror rA); L-ribocytidine-3′-phosphate(mirror rC); L-riboguanosine-3′-phosphate (mirror rG);L-ribouracil-3′-phosphate (mirror dU).

In some embodiments, modified ribonucleotides include modifieddeoxyribonucleotides, for example 5′OMe DNA(5-methyl-deoxyriboguanosine-3′-phosphate) which may be useful as anucleotide in the 5′ terminal position (position number 1); PACE(deoxyriboadenosine 3′ phosphonoacetate, deoxyribocytidine 3′phosphonoacetate, deoxyriboguanosine 3′ phosphonoacetate,deoxyribothymidine 3′ phosphonoacetate).

Modifications may be present in one or more strands of a nucleic acidmolecule disclosed herein, e.g., in the sense strand, the antisensestrand, or both strands. In certain embodiments, the antisense strandmay include modifications and the sense strand my only includeunmodified RNA.

Nucleobases

Nucleobases of the nucleic acid disclosed herein may include unmodifiedribonucleotides (purines and pyrimidines) such as adenine, guanine,cytosine, uracil. The nucleobases in one or both strands can be modifiedwith natural and synthetic nucleobases such as, thymine, xanthine,hypoxanthine, inosine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, any “universal base” nucleotides;2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, deazapurines, heterocyclic substitutedanalogs of purines and pyrimidines, e.g., aminoethyoxy phenoxazine,derivatives of purines and pyrimidines (e.g., 1-alkyl-, 1-alkenyl-,heteroaromatic- and 1-alkynyl derivatives) and tautomers thereof,8-oxo-N6-methyladenine, 7-diazaxanthine, 5-methylcytosine,5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl) cytosine and4,4-ethanocytosine). Other examples of suitable bases includenon-purinyl and non-pyrimidinyl bases such as 2-aminopyridine andtriazines.

Sugar Moieties

Sugar moieties in a nucleic acid disclosed herein may include2′-hydroxyl-pentofuranosyl sugar moiety without any modification.Alternatively, sugar moieties can be modified such as,2′-deoxy-pentofuranosyl sugar moiety, D-ribose, hexose, modification atthe 2′ position of the pentofuranosyl sugar moiety such as 2′-O-alkyl(including 2′OMe and 2′-O-ethyl), i.e., 2′-alkoxy, 2′-amino, 2′-O-allyl,2′-S-alkyl, 2′-halogen (including 2′-fluoro, chloro, and bromo),2′-methoxyethoxy, 2′-O-methoxyethyl, 2′-O-2-methoxyethyl, 2′-allyloxy(—OCH₂CH═CH₂), 2′-propargyl, 2′-propyl, ethynyl, ethenyl, propenyl, CF,cyano, imidazole, carboxylate, thioate, C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl, OCF₃, OCN, O-, S-, orN-alkyl; O-, S, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂, N₃;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino orsubstituted silyl, as, among others, for example as described inEuropean patents EP0586520 or EP0618925.

Alkyl group includes saturated aliphatic groups, includingstraight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups(isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups(cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkylsubstituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.In certain embodiments, a straight chain or branched chain alkyl has sixor fewer carbon atoms in its backbone (e.g., C₁-C₆ for straight chain,C₃-C₆ for branched chain), and more preferably four or fewer. Likewise,preferred cycloalkyls may have from three to eight carbon atoms in theirring structure, and more preferably have five or six carbons in the ringstructure. The term C₁-C₆ includes alkyl groups containing one to sixcarbon atoms. The alkyl group can be substituted alkyl group such asalkyl moieties having substituents replacing a hydrogen on one or morecarbons of the hydrocarbon backbone. Such substituents can include, forexample, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moiety.

Alkoxy group includes substituted and unsubstituted alkyl, alkenyl, andalkynyl groups covalently linked to an oxygen atom. Examples of alkoxygroups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, andpentoxy groups. Examples of substituted alkoxy groups includehalogenated alkoxy groups. The alkoxy groups can be substituted withgroups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties. Examples ofhalogen substituted alkoxy groups include, but are not limited to,fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy,dichloromethoxy, trichloromethoxy, etc.

In some embodiments, the pentafuranosyl ring may be replaced withacyclic derivatives lacking the C₂′-C₃′-bond of the pentafuronosyl ring.For example, acyclonucleotides may substitute a 2-hydroxyethoxymethylgroup for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs.

Halogens include fluorine, bromine, chlorine, iodine.

Backbone

The nucleoside subunits of the nucleic acid disclosed herein may belinked to each other by a phosphodiester bond. The phosphodiester bondmay be optionally substituted with other linkages. For example,phosphorothioate, thiophosphate-D-ribose entities, triester, thioate,2′-5′ bridged backbone (may also be referred to as 5′-2′ or2′-5′-nucleotide or 2′-5′-ribonucleotide), PACE, 3′(or -5′)-deoxy-3′(or-5′)-thio-phosphorothioate, phosphorodithioate, phosphoroselenates,3′(or -5′)-deoxy phosphinates, borano phosphates, 3′(or -5′)-deoxy-3′(or5′-)-amino phosphoramidates, hydrogen phosphonates, phosphonates, boranophosphate esters, phosphoramidates, alkyl or aryl phosphonates andphosphotriester modifications such as alkylphosphotriesters,phosphotriester phosphorus linkages, 5′-ethoxyphosphodiester,P-alkyloxyphosphotriester, methylphosphonate, and nonphosphoruscontaining linkages for example, carbonate, carbamate, silyl, sulfur,sulfonate, sulfonamide, formacetal, thioformacetyl, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino linkages.

Nucleic acid molecules disclosed herein may include a peptide nucleicacid (PNA) backbone. The PNA backbone includes repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The variousbases such as purine, pyrimidine, natural and synthetic bases are linkedto the backbone by methylene carbonyl bonds.

Terminal Phosphates

Modifications can be made at terminal phosphate groups. Non-limitingexamples of different stabilization chemistries can be used, e.g., tostabilize the 3′-end of nucleic acid sequences, including(3′-3′)-inverted deoxyribose; deoxyribonucleotide;(5′-3′)-3′-deoxyribonucleotide; (5′-3′)-ribonucleotide;(5′-3′)-3′-O-methyl ribonucleotide; 3′-glyceryl;(3′-5′)-3′-deoxyribonucleotide; (3′-3′)-deoxyribonucleotide;(5′-2′)-deoxyribonucleotide; and (5-3′)-dideoxyribonucleotide. Inaddition, unmodified backbone chemistries can be combined with one ormore different backbone modifications described herein.

Exemplary chemically modified terminal phosphate groups include thoseshown below:

Conjugates

Modified nucleotides and nucleic acid molecules (e.g., siNA molecules)as provided herein may include conjugates, for example, a conjugatecovalently attached to the chemically-modified nucleic acid molecule.Non-limiting examples of conjugates include conjugates and ligandsdescribed in Vargeese et al., U.S. Ser. No. 10/427,160. The conjugatemay be covalently attached to a nucleic acid molecule (such as an siNAmolecule) via a biodegradable linker. The conjugate molecule may beattached at the 3′-end of either the sense strand, the antisense strand,or both strands of the chemically-modified nucleic acid molecule. Theconjugate molecule may be attached at the 5′-end of either the sensestrand, the antisense strand, or both strands of the chemically-modifiednucleic acid molecule. The conjugate molecule may be attached both the3′-end and 5′-end of either the sense strand, the antisense strand, orboth strands of the chemically-modified nucleic acid molecule, or anycombination thereof. In one embodiment, a conjugate molecule may includea molecule that facilitates delivery of a chemically-modified nucleicacid molecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiednucleic acid molecule is a polyethylene glycol, human serum albumin, ora ligand for a cellular receptor that can mediate cellular uptake.Examples of specific conjugate molecules contemplated by the instantdescription that can be attached to chemically-modified nucleic acidmolecules are described in Vargeese et al., U.S. Ser. No. 10/201,394.

Linkers

A nucleic acid molecule provided herein (e.g., an siNA) may include anucleotide, non-nucleotide, or mixed nucleotide/non-nucleotide linkerthat joins the sense region of the nucleic acid to the antisense regionof the nucleic acid. A nucleotide linker can be a linker of >2nucleotides in length, for example about 3, 4, 5, 6, 7, 8, 9, or 10nucleotides in length. The nucleotide linker can be a nucleic acidaptamer. By “aptamer” or “nucleic acid aptamer” as used herein refers toa nucleic acid molecule that binds specifically to a target moleculewherein the nucleic acid molecule has sequence that includes a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule (such as hsp47 mRNA) where the target molecule does notnaturally bind to a nucleic acid. For example, the aptamer can be usedto bind to a ligand-binding domain of a protein, thereby preventinginteraction of the naturally occurring ligand with the protein. This isa non-limiting example and those in the art will recognize that otherembodiments can be readily generated using techniques generally known inthe art. See e.g., Gold et al.; 1995, Annu. Rev. Biochem., 64, 763;Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol.Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74, 27; Hermann and Patel,2000, Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45,1628.

A non-nucleotide linker may include an abasic nucleotide, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, orother polymeric compounds (e.g. polyethylene glycols such as thosehaving between 2 and 100 ethylene glycol units). Specific examplesinclude those described by Seela and Kaiser, Nucleic Acids Res. 1990,18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload and Schepartz, J.Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz, J. Am. Chem.Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993, 21:2585 andBiochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res. 1990,18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschkeet al., Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991,30:9914; Arnold et al., WO8902439; Usman et al., WO9506731; Dudycz etal., WO9511910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991,113:4000.

5′-Ends, 3′-Ends, and Overhangs

Nucleic acid molecules disclosed herein (e.g., siNA molecules) may beblunt-ended on both sides, have overhangs on both sides, or acombination of blunt and overhang ends. Overhangs may occur on eitherthe 5′- or 3′-end of the sense or antisense strand.

5′- and/or 3′-ends of double-stranded nucleic acid molecules (e.g.,siNA) may be blunt ended or have an overhang. The 5′-end may be bluntended and the 3′-end has an overhang in either the sense strand or theantisense strand. In other embodiments, the 3′-end may be blunt endedand the 5′-end has an overhang in either the sense strand or theantisense strand. In yet other embodiments, both the 5′- and 3′-end areblunt ended or both the 5′- and 3′-ends have overhangs.

The 5′- and/or 3′-end of one or both strands of the nucleic acid mayinclude a free hydroxyl group. The 5′- and/or 3′-end of any nucleic acidmolecule strand may be modified to include a chemical modification. Suchmodification may stabilize nucleic acid molecules, e.g., the 3′-end mayhave increased stability due to the presence of the nucleic acidmolecule modification. Examples of end modifications (e.g., terminalcaps) include, but are not limited to, abasic, deoxy abasic, inverted(deoxy) abasic, glyceryl, dinucleotide, acyclic nucleotide, amino,fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate,C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl,OCF₃, OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂, N₃; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino or substituted silyl, as, among others, described inEuropean patents EP586520 and EP618925 and other modifications disclosedherein.

Nucleic acid molecules include those with blunt ends, i.e., ends that donot include any overhanging nucleotides. A nucleic acid molecule caninclude one or more blunt ends. The blunt ended nucleic acid moleculehas a number of base pairs equal to the number of nucleotides present ineach strand of the nucleic acid molecule. The nucleic acid molecule caninclude one blunt end, for example where the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. Nucleic acid molecule may include one blunt end, forexample where the 3′-end of the antisense strand and the 5′-end of thesense strand do not have any overhanging nucleotides. A nucleic acidmolecule may include two blunt ends, for example where the 3′-end of theantisense strand and the 5′-end of the sense strand as well as the5′-end of the antisense strand and 3′-end of the sense strand do nothave any overhanging nucleotides. Other nucleotides present in a bluntended nucleic acid molecule can include, for example, mismatches,bulges, loops, or wobble base pairs to modulate the activity of thenucleic acid molecule to mediate RNA interference.

In certain embodiments of the nucleic acid molecules (e.g., siNAmolecules) provided herein, at least one end of the molecule has anoverhang of at least one nucleotide (for example one to eight overhangnucleotides). For example, one or both strands of a double-strandednucleic acid molecule disclosed herein may have an overhang at the5′-end or at the 3′-end or both. An overhang may be present at either orboth the sense strand and antisense strand of the nucleic acid molecule.The length of the overhang may be as little as one nucleotide and aslong as one to eight or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7 or 8nucleotides; in some preferred embodiments an overhang is 2, 3, 4, 5, 6,7 or 8 nucleotides; for example an overhang may be 2 nucleotides. Thenucleotide(s) forming the overhang may include deoxyribonucleotide(s),ribonucleotide(s), natural and non-natural nucleobases or any nucleotidemodified in the sugar, base or phosphate group such as disclosed herein.A double-stranded nucleic acid molecule may have both 5′- and3′-overhangs. The overhangs at the 5′- and 3′-end may be of differentlengths. An overhang may include at least one nucleic acid modificationwhich may be deoxyribonucleotide. One or more deoxyribonucleotides maybe at the 5′-terminal. The 3′-end of the respective counter-strand ofthe nucleic acid molecule may not have an overhang, more preferably nota deoxyribonucleotide overhang. The one or more deoxyribonucleotide maybe at the 3′-terminal. The 5′-end of the respective counter-strand ofthe dsRNA may not have an overhang, more preferably not adeoxyribonucleotide overhang. The overhang in either the 5′- or the3′-end of a strand may be one to eight (e.g., about 1, 2, 3, 4, 5, 6, 7or 8) unpaired nucleotides, preferably, the overhang is two to threeunpaired nucleotides; more preferably two unpaired nucleotides. Nucleicacid molecules may include duplex nucleic acid molecules withoverhanging ends of about 1 to about 20 (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 1, 15, 16, 17, 18, 19 or 20); preferably one toeight (e.g., about 1, 2, 3, 4, 5, 6, 7 or 8) nucleotides, for example,about 21-nucleotide duplexes with about 19 base pairs and 3′-terminalmononucleotide, dinucleotide, or trinucleotide overhangs. Nucleic acidmolecules herein may include duplex nucleic acid molecules with bluntends, where both ends are blunt, or alternatively, where one of the endsis blunt. Nucleic acid molecules disclosed herein can include one ormore blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended nucleic acid moleculehas a number of base pairs equal to the number of nucleotides present ineach strand of the nucleic acid molecule. The nucleic acid molecule mayinclude one blunt end, for example where the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. The nucleic acid molecule may include one blunt end, forexample where the 3′-end of the antisense strand and the 5′-end of thesense strand do not have any overhanging nucleotides. A nucleic acidmolecule may include two blunt ends, for example where the 3′-end of theantisense strand and the 5′-end of the sense strand as well as the5′-end of the antisense strand and 3′-end of the sense strand do nothave any overhanging nucleotides. In certain preferred embodiments thenucleic acid compounds are blunt ended. Other nucleotides present in ablunt ended siNA molecule can include, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the nucleic acidmolecule to mediate RNA interference.

In many embodiments one or more, or all, of the overhang nucleotides ofa nucleic acid molecule (e.g., a siNA molecule) as described hereinincludes are modified such as described herein; for example one or more,or all, of the nucleotides may be 2′-deoxynucleotides.

Amount, Location and Patterns of Modifications

Nucleic acid molecules (e.g., siNA molecules) disclosed herein mayinclude modified nucleotides as a percentage of the total number ofnucleotides present in the nucleic acid molecule. As such, a nucleicacid molecule may include about 5% to about 100% modified nucleotides(e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides). Theactual percentage of modified nucleotides present in a given nucleicacid molecule will depend on the total number of nucleotides present inthe nucleic acid. If the nucleic acid molecule is single-stranded, thepercent modification can be based upon the total number of nucleotidespresent in the single-stranded nucleic acid molecule. Likewise, if thenucleic acid molecule is double-stranded, the percent modification canbe based upon the total number of nucleotides present in the sensestrand, antisense strand, or both the sense and antisense strands.

Nucleic acid molecules disclosed herein may include unmodified RNA as apercentage of the total nucleotides in the nucleic acid molecule. Assuch, a nucleic acid molecule may include about 5% to about 100%modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) of totalnucleotides present in a nucleic acid molecule.

A nucleic acid molecule (e.g., an siNA molecule) may include a sensestrand that includes about one to about five, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′OMe,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand includes about one toabout five or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′OMe,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. A nucleic acid molecule may includeabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense nucleic acid strand are chemically-modifiedwith 2′-deoxy, 2′OMe and/or 2′-deoxy-2′-fluoro nucleotides, with orwithout about one to about five or more, for example about 1, 2, 3, 4,5, or more phosphorothioate internucleotide linkages and/or a terminalcap molecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends,being present in the same or different strand.

A nucleic acid molecule may include about one to about five or more(specifically about 1, 2, 3, 4, 5, or more) phosphorothioateinternucleotide linkages in each strand of the nucleic acid molecule.

A nucleic acid molecule may include 2′-5′ internucleotide linkages, forexample at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of oneor both nucleic acid sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both nucleic acid sequence strands, for example, about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotidelinkage of a pyrimidine nucleotide in one or both strands of the siNAmolecule can include a 2′-5′ internucleotide linkage, or about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkage ofa purine nucleotide in one or both strands of the siNA molecule caninclude a 2′-5′ internucleotide linkage.

A chemically-modified short interfering nucleic acid (siNA) molecule mayinclude an antisense region, wherein any (e.g., one or more or all)pyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides).

A chemically-modified short interfering nucleic acid (siNA) molecule mayinclude an antisense region, wherein any (e.g., one or more or all)pyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′OMe purine nucleotides(e.g., wherein all purine nucleotides are 2′OMe purine nucleotides oralternately a plurality of purine nucleotides are 2′OMe purinenucleotides).

A chemically-modified siNA molecule capable of mediating RNAinterference (RNAi) against hsp47 inside a cell or reconstituted invitro system may include a sense region, wherein one or more pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and one or more purine nucleotides present in the sense region are2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are2′-deoxy purine nucleotides or alternately a plurality of purinenucleotides are 2′-deoxy purine nucleotides), and an antisense region,wherein one or more pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and one or more purine nucleotides present inthe antisense region are 2′OMe purine nucleotides (e.g., wherein allpurine nucleotides are 2′OMe purine nucleotides or alternately aplurality of purine nucleotides are 2′OMe purine nucleotides). The senseregion and/or the antisense region can have a terminal cap modification,such as any modification that is optionally present at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense and/or antisensesequence. The sense and/or antisense region can optionally furtherinclude a 3′-terminal nucleotide overhang having about 1 to about 4(e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further include one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. The purine nucleotides in the sense region mayalternatively be 2′OMe purine nucleotides (e.g., wherein all purinenucleotides are 2′OMe purine nucleotides or alternately a plurality ofpurine nucleotides are 2′OMe purine nucleotides) and one or more purinenucleotides present in the antisense region are 2′OMe purine nucleotides(e.g., wherein all purine nucleotides are 2′OMe purine nucleotides oralternately a plurality of purine nucleotides are 2′OMe purinenucleotides). One or more purine nucleotides in the sense region mayalternatively be purine ribonucleotides (e.g., wherein all purinenucleotides are purine ribonucleotides or alternately a plurality ofpurine nucleotides are purine ribonucleotides) and any purinenucleotides present in the antisense region are 2′OMe purine nucleotides(e.g., wherein all purine nucleotides are 2′OMe purine nucleotides oralternately a plurality of purine nucleotides are 2′OMe purinenucleotides). One or more purine nucleotides in the sense region and/orpresent in the antisense region may alternatively selected from thegroup consisting of 2′-deoxy nucleotides, LNA nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′OMe nucleotides(e.g., wherein all purine nucleotides are selected from the groupconsisting of 2′-deoxy nucleotides, LNA nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′OMe nucleotides or alternately aplurality of purine nucleotides are selected from the group consistingof 2′-deoxy nucleotides, LNA nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′OMe nucleotides).

In some embodiments, a nucleic acid molecule (e.g., a siNA molecule) asdescribed herein includes a modified nucleotide (for example onemodified nucleotide) in the antisense strand; for example in position 6or position 7 of the antisense strand.

Modification Patterns and Alternating Modifications

Nucleic acid molecules (e.g., siNA molecules) provided herein may havepatterns of modified and unmodified nucleic acids. A pattern ofmodification of the nucleotides in a contiguous stretch of nucleotidesmay be a modification contained within a single nucleotide or group ofnucleotides that are covalently linked to each other via standardphosphodiester bonds or, at least partially, through phosphorothioatebonds. Accordingly, a “pattern” as contemplated herein, does notnecessarily need to involve repeating units, although it may. Examplesof modification patterns that may be used in conjunction with thenucleic acid molecules (e.g., siNA molecules) provided herein includethose disclosed in Giese, U.S. Pat. No. 7,452,987. For example, nucleicacid molecules (e.g., siNA molecules) provided herein include those withmodification patterns such as, similar to, or the same as, the patternsshown diagrammatically in FIG. 2 of U.S. Pat. No. 7,452,987.

A modified nucleotide or group of modified nucleotides may be at the5′-end or 3′-end of the sense or antisense strand, a flanking nucleotideor group of nucleotides is arrayed on both sides of the modifiednucleotide or group, where the flanking nucleotide or group either isunmodified or does not have the same modification of the precedingnucleotide or group of nucleotides. The flanking nucleotide or group ofnucleotides may, however, have a different modification. This sequenceof modified nucleotide or group of modified nucleotides, respectively,and unmodified or differently modified nucleotide or group of unmodifiedor differently modified nucleotides may be repeated one or more times.

In some patterns, the 5′-terminal nucleotide of a strand is a modifiednucleotide while in other patterns the 5′-terminal nucleotide of astrand is an unmodified nucleotide. In some patterns, the 5′-end of astrand starts with a group of modified nucleotides while in otherpatterns, the 5′-terminal end is an unmodified group of nucleotides.This pattern may be either on the first stretch or the second stretch ofthe nucleic acid molecule or on both.

Modified nucleotides of one strand of the nucleic acid molecule may becomplementary in position to the modified or unmodified nucleotides orgroups of nucleotides of the other strand.

There may be a phase shift between modifications or patterns ofmodifications on one strand relative to the pattern of modification ofthe other strand such that the modification groups do not overlap. Inone instance, the shift is such that the modified group of nucleotidesof the sense strand corresponds to the unmodified group of nucleotidesof the antisense strand and vice versa.

There may be a partial shift of the pattern of modification such thatthe modified groups overlap. The groups of modified nucleotides in anygiven strand may optionally be the same length, but may be of differentlengths. Similarly, groups of unmodified nucleotides in any given strandmay optionally be the same length, or of different lengths.

In some patterns, the second (penultimate) nucleotide at the terminus ofthe strand, is an unmodified nucleotide or the beginning of group ofunmodified nucleotides. Preferably, this unmodified nucleotide orunmodified group of nucleotides is located at the 5′-end of the eitheror both the sense and antisense strands and even more preferably at theterminus of the sense strand. An unmodified nucleotide or unmodifiedgroup of nucleotide may be located at the 5′-end of the sense strand. Ina preferred embodiment the pattern consists of alternating singlemodified and unmodified nucleotides.

Some double-stranded nucleic acid molecules include a 2′OMe modifiednucleotide and a non-modified nucleotide, preferably a nucleotide whichis not 2′OMe modified, are incorporated on both strands in analternating fashion, resulting in a pattern of alternating 2′OMemodified nucleotides and nucleotides that are either unmodified or atleast do not include a 2′OMe modification. In certain embodiments, thesame sequence of 2′OMe modification and non-modification exists on thesecond strand; in other embodiments the alternating 2′OMe modifiednucleotides are only present in the sense strand and are not present inthe antisense strand; and in yet other embodiments the alternating 2′OMemodified nucleotides are only present in the sense strand and are notpresent in the antisense strand. In certain embodiments, there is aphase shift between the two strands such that the 2′OMe modifiednucleotide on the first strand base pairs with a non-modifiednucleotide(s) on the second strand and vice versa. This particulararrangement, i.e. base pairing of 2′OMe modified and non-modifiednucleotide(s) on both strands is particularly preferred in certainembodiments. In certain embodiments, the pattern of alternating 2′OMemodified nucleotides exists throughout the entire nucleic acid molecule;or the entire duplex region. In other embodiments the pattern ofalternating 2′OMe modified nucleotides exists only in a portion of thenucleic acid; or the entire duplex region.

In “phase shift” patterns, it may be preferred if the antisense strandstarts with a 2′OMe modified nucleotide at the 5′-end wherebyconsequently the second nucleotide is non-modified, the third, fifth,seventh and so on nucleotides are thus again 2′OMe modified whereas thesecond, fourth, sixth, eighth and the like nucleotides are non-modifiednucleotides.

Modification Locations and Patterns

While exemplary patterns are provided in more detail below, allpermutations of patterns with of all possible characteristics of thenucleic acid molecules disclosed herein and those known in the art arecontemplated (e.g., characteristics include, but are not limited to,length of sense strand, length of antisense strand, length of duplexregion, length of overhang, whether one or both ends of adouble-stranded nucleic acid molecule is blunt or has an overhang,location of modified nucleic acid, number of modified nucleic acids,types of modifications, whether a double overhang nucleic acid moleculehas the same or different number of nucleotides on the overhang of eachside, whether a one or more than one type of modification is used in anucleic acid molecule, and number of contiguous modified/unmodifiednucleotides). With respect to all detailed examples provided below,while the duplex region is shown to be 19 nucleotides, the nucleic acidmolecules provided herein can have a duplex region ranging from 1 to 49nucleotides in length as each strand of a duplex region canindependently be 17-49 nucleotides in length Exemplary patterns areprovided herein.

Nucleic acid molecules may have a blunt end (when n is 0) on both endsthat include a single or contiguous set of modified nucleic acids. Themodified nucleic acid may be located at any position along either thesense or antisense strand. Nucleic acid molecules may include a group ofabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 contiguous modifiednucleotides. Modified nucleic acids may make up 1%, 2%, 3%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98% or 100% of a nucleic acid strand. Modifiednucleic acids of the examples immediately below may be in the sensestrand only, the antisense strand only, or in both the sense andantisense strand.

General nucleic acid patterns are shown below where X=sense strandnucleotide in the duplex region; X_(a)=5′-overhang nucleotide in thesense strand; X_(b)=3′-overhang nucleotide in the sense strand;Y=antisense strand nucleotide in the duplex region; Y_(a)=3′-overhangnucleotide in the antisense strand; Y_(b)=5′-overhang nucleotide in theantisense strand; and M=a modified nucleotide in the duplex region. Eacha and b are independently 0 to 8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7 or 8).Each X, Y, a and b are independently modified or unmodified. The senseand antisense strands can are each independently 17-49 nucleotides inlength. The examples provided below have a duplex region of 19nucleotides; however, nucleic acid molecules disclosed herein can have aduplex region anywhere between 17 and 49 nucleotides and where eachstrand is independently between 17 and 49 nucleotides in length.

5′ X_(a)XXXXXXXXXXXXXXXXXXXX_(b) 3′ Y_(b)YYYYYYYYYYYYYYYYYYYY_(a)

Further exemplary nucleic acid molecule patterns are shown below whereX=unmodified sense strand nucleotides; x=an unmodified overhangnucleotide in the sense strand; Y=unmodified antisense strandnucleotides; y=an unmodified overhang nucleotide in the antisensestrand; and M=a modified nucleotide. The sense and antisense strands canare each independently 17-49 nucleotides in length. The examplesprovided below have a duplex region of 19 nucleotides; however, nucleicacid molecules disclosed herein can have a duplex region anywherebetween 17 and 49 nucleotides and where each strand is independentlybetween 17 and 49 nucleotides in length.

5′ M _(n)XXXXXXXXXMXXXXXXXXXM _(n) 3′ M _(n)YYYYYYYYYYYYYYYYYYYM _(n) 5′XXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYYMYYYYYYYYY 5′ XXXXXXXXMMXXXXXXXXX 3′YYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYMMYYYYYYYYY 5′XXXXXXXXXMXXXXXXXXX 3′ YYYYYYYYYMYYYYYYYYY 5′ XXXXXMXXXXXXXXXXXXX 3′YYYYYYYYYMYYYYYYYYY 5′ MXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYYYYYMYYYYYY 5′XXXXXXXXXXXXXXXXXXM 3′ YYYYYMYYYYYYYYYYYYY 5′ XXXXXXXXXMXXXXXXXX 3′MYYYYYYYYYYYYYYYYY 5′ XXXXXXXMXXXXXXXXXX 3′ YYYYYYYYYYYYYYYYYM 5′XXXXXXXXXXXXXMXXXX 3′ MYYYYYYYYYYYYYYYYY 5′ MMMMMMMMMMMMMMMMMM 3′MMMMMMMMMMMMMMMMMM

Nucleic acid molecules may have blunt ends on both ends with alternatingmodified nucleic acids. The modified nucleic acids may be located at anyposition along either the sense or antisense strand.

5′ MXMXMXMXMXMXMXMXMXM 3′ YMYMYMYMYMYMYMYMYMY 5′ XMXMXMXMXMXMXMXMXMX 3′MYMYMYMYMYMYMYMYMYM 5′ MMXMMXMMXMMXMMXMMXM 3′ YMMYMMYMMYMMYMMYMMY 5′XMMXMMXMMXMMXMMXMMX 3′ MMYMMYMMYMMYMMYMMYM 5′ MMMXMMMXMMMXMMMXMMM 3′YMMMYMMMYMMMYMMMYMM 5′ XMMMXMMMXMMMXMMMXMM 3′ MMMYMMMYMMMYMMMYMMM

Nucleic acid molecules with a blunt 5′-end and 3′-end overhang end witha single modified nucleic acid.

Nucleic acid molecules with a 5′-end overhang and a blunt 3′-end with asingle modified nucleic acid.

Nucleic acid molecules with overhangs on both ends and all overhangs aremodified nucleic acids. In the pattern immediately below, M is n numberof modified nucleic acids, where n is an integer from 0 to 8 (i.e., 0,1, 2, 3, 4, 5, 6, 7 and 8).

5′  XXXXXXXXXXXXXXXXXXXM 3′ MYYYYYYYYYYYYYYYYYYY

Nucleic acid molecules with overhangs on both ends and some overhangnucleotides are modified nucleotides. In the patterns immediately below,M is n number of modified nucleotides, x is n number of unmodifiedoverhang nucleotides in the sense strand, y is n number of unmodifiedoverhang nucleotides in the antisense strand, where each n isindependently an integer from 0 to 8 (i.e., 0, 1, 2, 3, 4, 5, 6, 7 and8), and where each overhang is maximum of 20 nucleotides; preferably amaximum of 8 nucleotides (modified and/or unmodified).

5′  XXXXXXXXXXXXXXXXXXXM 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMx 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMxM 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMxMx 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMxMxM 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMxMxMx 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMxMxMxM 3′ yYYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMxMxMxMx 3′ yYYYYYYYYYYYYYYYYYYY 5′MXXXXXXXXXXXXXXXXXXX 3′  YYYYYYYYYYYYYYYYYYYy 5′ xMXXXXXXXXXXXXXXXXXXX3′   YYYYYYYYYYYYYYYYYYYy 5′ MxMXXXXXXXXXXXXXXXXXXX 3′   YYYYYYYYYYYYYYYYYYYy 5′ xMxMXXXXXXXXXXXXXXXXXXX 3′    YYYYYYYYYYYYYYYYYYYy 5′ MxMxMXXXXXXXXXXXXXXXXXXX 3′     YYYYYYYYYYYYYYYYYYYy 5′ xMxMxMXXXXXXXXXXXXXXXXXXX 3′      YYYYYYYYYYYYYYYYYYYy 5′ MxMxMxMXXXXXXXXXXXXXXXXXXX 3′       YYYYYYYYYYYYYYYYYYYy 5′ xMxMxMxMXXXXXXXXXXXXXXXXXXX 3′        YYYYYYYYYYYYYYYYYYYy 5′ xXXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYYYYYYYYYYYYM 5′ xXXXXXXXXXXXXXXXXXXX 3′  YYYYYYYYYYYYYYYYYYYMy5′ xXXXXXXXXXXXXXXXXXXX 3′  YYYYYYYYYYYYYYYYYYYMyM 5′xXXXXXXXXXXXXXXXXXXX 3′  YYYYYYYYYYYYYYYYYYYMyMy 5′ xXXXXXXXXXXXXXXXXXXX3′  YYYYYYYYYYYYYYYYYYYMyMyM 5′ xXXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYYYYYYYYYYYYMyMyMy 5′ xXXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYYYYYYYYYYYYMyMyMyM 5′ xXXXXXXXXXXXXXXXXXXX 3′ YYYYYYYYYYYYYYYYYYYMyMyMyMy 5′  XXXXXXXXXXXXXXXXXXXx 3′MYYYYYYYYYYYYYYYYYYY 5′   XXXXXXXXXXXXXXXXXXXx 3′ yMYYYYYYYYYYYYYYYYYYY5′    XXXXXXXXXXXXXXXXXXXx 3′ MyMYYYYYYYYYYYYYYYYYYY 5′    XXXXXXXXXXXXXXXXXXXx 3′ yMyMYYYYYYYYYYYYYYYYYYY 5′     XXXXXXXXXXXXXXXXXXXx 3′ MyMyMYYYYYYYYYYYYYYYYYYY 5′      XXXXXXXXXXXXXXXXXXXx 3′ yMyMyMYYYYYYYYYYYYYYYYYYY 5′       XXXXXXXXXXXXXXXXXXXx 3′ MyMyMyMYYYYYYYYYYYYYYYYYYY 5′        XXXXXXXXXXXXXXXXXXXx 3′ yMyMyMyMYYYYYYYYYYYYYYYYYYY

Modified nucleotides at the 3′-end of the sense region.

5′ XXXXXXXXXXXXXXXXXXXM 3′ YYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMM3′ YYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMMM 3′ YYYYYYYYYYYYYYYYYYY5′ XXXXXXXXXXXXXXXXXXXMMMM 3′ YYYYYYYYYYYYYYYYYYY 5′XXXXXXXXXXXXXXXXXXXMMMMM 3′ YYYYYYYYYYYYYYYYYYY 5′XXXXXXXXXXXXXXXXXXXMMMMMM 3′ YYYYYYYYYYYYYYYYYYY 5′XXXXXXXXXXXXXXXXXXXMMMMMMMM 3′ YYYYYYYYYYYYYYYYYYY 5′XXXXXXXXXXXXXXXXXXXMMMMMMMM 3′ YYYYYYYYYYYYYYYYYYY

Overhang at the 5′-end of the sense region.

5′ MXXXXXXXXXXXXXXXXXXX 3′  YYYYYYYYYYYYYYYYYYY 5′ MMXXXXXXXXXXXXXXXXXXX3′   YYYYYYYYYYYYYYYYYYY 5′ MMMXXXXXXXXXXXXXXXXXXX 3′   YYYYYYYYYYYYYYYYYYY 5′ MMMMXXXXXXXXXXXXXXXXXXX 3′    YYYYYYYYYYYYYYYYYYY 5′ MMMMMXXXXXXXXXXXXXXXXXXX 3′     YYYYYYYYYYYYYYYYYYY 5′ MMMMMMXXXXXXXXXXXXXXXXXXX 3′      YYYYYYYYYYYYYYYYYYY 5′ MMMMMMMXXXXXXXXXXXXXXXXXXX 3′       YYYYYYYYYYYYYYYYYYY 5′ MMMMMMMMXXXXXXXXXXXXXXXXXXX 3′        YYYYYYYYYYYYYYYYYYY

Overhang at the 3′-end of the antisense region.

5′  XXXXXXXXXXXXXXXXXXX 3′ MYYYYYYYYYYYYYYYYYYY 5′   XXXXXXXXXXXXXXXXXXX3′ MMYYYYYYYYYYYYYYYYYYY 5′    XXXXXXXXXXXXXXXXXXX 3′MMMYYYYYYYYYYYYYYYYYYY 5′     XXXXXXXXXXXXXXXXXXX 3′MMMMYYYYYYYYYYYYYYYYYYY 5′      XXXXXXXXXXXXXXXXXXX 3′MMMMMYYYYYYYYYYYYYYYYYYY 5′       XXXXXXXXXXXXXXXXXXX 3′MMMMMMYYYYYYYYYYYYYYYYYYY 5′        XXXXXXXXXXXXXXXXXXX 3′MMMMMMMYYYYYYYYYYYYYYYYYYY 5′         XXXXXXXXXXXXXXXXXXX 3′MMMMMMMMYYYYYYYYYYYYYYYYYYY

Modified nucleotide(s) within the sense region

5′ XXXXXXXXXMXXXXXXXXX 3′ YYYYYYYYYYYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXX 3′YYYYYYYYYMYYYYYYYYY 5′ XXXXXXXXXXXXXXXXXXXMM 3′ YYYYYYYYYYYYYYYYYYY 5′  XXXXXXXXXXXXXXXXXXX 3′ MMYYYYYYYYYYYYYYYYYYY

Exemplary nucleic acid molecules are provided below along with theequivalent general structure in line with the symbols used above:

siHSP47-C siRNA to human and rat hsp47 having a 19 nucleotide (i.e.,19mer) duplex region and modified 2 nucleotide (i.e., deoxynucleotide)overhangs at the 3′-ends of the sense and antisense strands.

5′     GGACAGGCCUCUACAACUAdTdT 3′ (SEQ ID NO: 5) 3′dTdTCCUGUCCGGAGAUGUUGAU 5′ (SEQ ID NO: 6) 5′   XXXXXXXXXXXXXXXXXXXMM 3′MMYYYYYYYYYYYYYYYYYYY

siHSP47-Cd siRNA to human and rat hsp47 having a 25-mer duplex region, a2 nucleotide overhang at the 3′-end of the antisense strand and 2modified nucleotides at the 5′-terminal and penultimate positions of thesense strand.

5′   GGACAGGCCUCUACAACUACUACdGdA 3′ (SEQ ID NO: 9) 3′UUCCUGUCCGGAGAUGUUGAUGAUGCU 5′ (SEQ ID NO: 10) 5′  XXXXXXXXXXXXXXXXXXXXXXXMM 3′ 3′ yyYYYYYYYYYYYYYYYYYYYYYYYYY 5′

siHSP47-1 siRNA to human and rat hsp47 cDNA 719-737 having a 19-merduplex region, and modified 2 nucleotide (i.e., deoxynucleotide)overhangs at the 3′-ends of the sense and antisense strands.

5′     CAGGCCUCUACAACUACUAdTdT 3′ (SEQ ID NO: 13) 3′dTdTGUCCGGAGAUGUUGAUGAU 5′ (SEQ ID NO: 14) 5′   XXXXXXXXXXXXXXXXXXXMM 3′3′ MMYYYYYYYYYYYYYYYYYYY 5′

siHSP47-1d siRNA to human hsp47 cDNA 719-743 having a 25-mer with ablunt end at the 3′-end of the sense strand and a 2 nucleotide overhangat the 3′-end of the antisense strand, and 2 modified nucleotides at the5′-terminal and penultimate positions of the sense strand.

5′   CAGGCCUCUACAACUACUACGACdGdA 3′ (SEQ ID NO: 17) 3′UUGUCCGGAGAUGUUGAUGAUGCUGCU 5′ (SEQ ID NO: 2724) 5′  XXXXXXXXXXXXXXXXXXXXXXXMM 3′ 3′ yyYYYYYYYYYYYYYYYYYYYYYYYYY 5′

siHSP47-2 siRNA to human hsp47 cDNA 469-487 having a 19-mer duplexregion, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs atthe 3′-ends of the sense and antisense strands.

5′   GAGCACUCCAAGAUCAACUdTdT 3′ (SEQ ID NO: 21) 3′dTdTCUCGUGAGGUUCUAGUUGA 5′ (SEQ ID NO: 22) 5′   XXXXXXXXXXXXXXXXXXXMM 3′3′ MMYYYYYYYYYYYYYYYYYYY 5′

siHSP47-2d siRNA to human hsp47 cDNA 469-493 having a 25-mer duplexregion with a blunt end at the 3′-end of the sense strand and a 2nucleotide overhang at the 3′-end of the antisense strand, and 2modified nucleotides at the 5′-terminal and penultimate positions of thesense strand.

5′   GAGCACUCCAAGAUCAACUUCCGdCdG 3′ (SEQ ID NO: 25) 3′UUCUCGUGAGGUUCUAGUUGAAGGCGC 5′ (SEQ ID NO: 24) 5′  XXXXXXXXXXXXXXXXXXXXXXXMM 3′ 3′ yyYYYYYYYYYYYYYYYYYYYYYYYYY 5′

siHSP47-2d rat siRNA to rat Gp46 cDNA 466-490 having a 25-mer duplexregion with a blunt end at the 3′-end of the sense strand and a 2nucleotide overhang at the 3′-end of the antisense strand, and 2modified nucleotides at the 5′-terminal and penultimate positions of thesense strand.

5′   GAACACUCCAAGAUCAACUUCCGdAdG 3′ (SEQ ID NO: 29) 3′UUCUUGUGAGGUUCUAGUUGAAGGCUC 5′ (SEQ ID NO: 28) 5′  XXXXXXXXXXXXXXXXXXXXXXXMM 3′ 3′ yyYYYYYYYYYYYYYYYYYYYYYYYYY 5′

siHSP47-3 siRNA to human hsp47 cDNA 980-998 having a 19-mer duplexregion, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs atthe 3′-ends of the sense and antisense strands.

5′   CTGAGGCCATTGACAAGAAdTdT 3′ (SEQ ID NO: 2725) 3′dTdTGACUCCGGUAACUGUUCUU 5′ (SEQ ID NO: 34) 5′   XXXXXXXXXXXXXXXXXXXMM 3′3′ MMYYYYYYYYYYYYYYYYYYY 5′

siHSP47-3d siRNA to human hsp47 cDNA 980-1004 having a 25-mer duplexregion with a blunt end at the 3′-end of the sense strand and a 2nucleotide overhang at the 3′-end of the antisense strand, and 2modified nucleotides at the 5′-terminal and penultimate positions of thesense strand.

5′   CTGAGGCCATTGACAAGAACAAGdGdC 3′ (SEQ ID NO: 2726) 3′UUGACUCCGGUAACUGUUCUUGUUCCG 5′ (SEQ ID NO: 38) 5′  XXXXXXXXXXXXXXXXXXXXXXXMM 3′ 3′ yyYYYYYYYYYYYYYYYYYYYYYYYYY 5′

siHSP47-4 siRNA to human hsp47 cDNA 735-753 having a 19-mer duplexregion, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs atthe 3′-ends of the sense and antisense strands.

5′   CUACGACGACGAGAAGGAAdTdT 3′ (SEQ ID NO: 41) 3′dTdTGAUGCUGCUGCUCUUCCUU 5′ (SEQ ID NO: 42) 5′   XXXXXXXXXXXXXXXXXXXMM 3′3′ MMYYYYYYYYYYYYYYYYYYY 5′

siHSP47-4d siRNA to human hsp47 cDNA 735-759 having a 25-mer duplexregion with a blunt end at the 3′-end of the sense strand and a 2nucleotide overhang at the 3′-end of the antisense strand, and 2modified nucleotides at the 5′-terminal and penultimate positions of thesense strand.

5′   CUACGACGACGAGAAGGAAAAGCdTdG 3′ (SEQ ID NO: 45) 3′UUGAUGCUGCUGCUCUUCCUUUUCGAC 5′ (SEQ ID NO: 2727) 5′  XXXXXXXXXXXXXXXXXXXXXXXMM 3′ 3′ yyYYYYYYYYYYYYYYYYYYYYYYYYY 5′

siHSP47-5 siRNA to human hsp47 cDNA 621-639 having a 19-mer duplexregion, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs atthe 3′-ends of the sense and antisense strands.

5′     GCCACACUGGGAUGAGAAAdTdT 3′ (SEQ ID NO: 49) 3′dTdTCGGUGUGACCCUACUCUUU 5′ (SEQ ID NO: 50) 5′     XXXXXXXXXXXXXXXXXXXMM3′ 3′   MMYYYYYYYYYYYYYYYYYYY 5′

siHSP47-6 siRNA to human hsp47 cDNA 446-464 having a 19-mer duplexregion, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs atthe 3′-ends of the sense and antisense strands.

5′     GCAGCAAGCAGCACUACAAdTdT 3′ (SEQ ID NO: 53) 3′dTdTCGUCGUUCGUCGUGAUGUU 5′ (SEQ ID NO: 55) 5′     XXXXXXXXXXXXXXXXXXXMM3′ 3′   MMYYYYYYYYYYYYYYYYYYY 5′siHSP47-7 siRNA to human hsp47 cDNA 692-710 having a 19-mer duplexregion, and modified 2 nucleotide (i.e., deoxynucleotide) overhangs atthe 3′-ends of the sense and antisense strands.

5′     CCGUGGGUGUCAUGAUGAUdTdT 3′ (SEQ ID NO: 57) 3′dTdTGGCACCCACAGUACUACUA 5′ (SEQ ID NO: 58) 5′     XXXXXXXXXXXXXXXXXXXMM3′ 3′   MMYYYYYYYYYYYYYYYYYYY 5′

Nicks and Gaps in Nucleic Acid Strands

Nucleic acid molecules (e.g., siNA molecules) provided herein may have astrand, preferably, the sense strand, that is nicked or gapped. As such,nucleic acid molecules may have three or more strand, for example, suchas a meroduplex RNA (mdRNA) disclosed in PCT/US07/081836. Nucleic acidmolecules with a nicked or gapped strand may be between about 1-49nucleotides, or may be RISC length (e.g., about 15 to 25 nucleotides) orDicer substrate length (e.g., about 25 to 30 nucleotides) such asdisclosed herein.

Nucleic acid molecules with three or more strands include, for example,an ‘A’ (antisense) strand, ‘S1’ (second) strand, and ‘S2’ (third) strandin which the ‘S1’ and ‘S2’ strands are complementary to and form basepairs with non-overlapping regions of the ‘A’ strand (e.g., an mdRNA canhave the form of A:S1S2). The S1, S2, or more strands together form whatis substantially similar to a sense strand to the ‘A’ antisense strand.The double-stranded region formed by the annealing of the ‘S1’ and ‘A’strands is distinct from and non-overlapping with the double-strandedregion formed by the annealing of the ‘S2’ and ‘A’ strands. A nucleicacid molecule (e.g., an siNA molecule) may be a “gapped” molecule,meaning a “gap” ranging from 0 nucleotides up to about 10 nucleotides(e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides). Preferably, thesense strand is gapped. In some embodiments, the A:S1 duplex isseparated from the A:S2 duplex by a gap resulting from at least oneunpaired nucleotide (up to about 10 unpaired nucleotides) in the ‘A’strand that is positioned between the A:S1 duplex and the A: S2 duplexand that is distinct from any one or more unpaired nucleotide at the3′-end of one or more of the ‘A’, ‘S1’, or ‘S2 strands. The A:S1 duplexmay be separated from the A:B2 duplex by a gap of zero nucleotides(i.e., a nick in which only a phosphodiester bond between twonucleotides is broken or missing in the polynucleotide molecule) betweenthe A:S1 duplex and the A:S2 duplex-which can also be referred to asnicked dsRNA (ndsRNA). For example, A:S1S2 may include a dsRNA having atleast two double-stranded regions that combined total about 14 basepairs to about 40 base pairs and the double-stranded regions areseparated by a gap of about 0 to about 10 nucleotides, optionally havingblunt ends, or A:S1S2 may include a dsRNA having at least twodouble-stranded regions separated by a gap of up to ten nucleotideswherein at least one of the double-stranded regions includes betweenabout five base pairs and thirteen base pairs.

Dicer Substrates

In certain embodiments, the nucleic acid molecules (e.g., siNAmolecules) provided herein may be a precursor “Dicer substrate”molecule, e.g., double-stranded nucleic acid, processed in vivo toproduce an active nucleic acid molecules, for example as described inRossi, US20050244858. In certain conditions and situations, it has beenfound that these relatively longer dsRNA siNA species, e.g., of fromabout 25 to about 30 nucleotides, can give unexpectedly effectiveresults in terms of potency and duration of action. Without wishing tobe bound by any particular theory, it is thought that the longer dsRNAspecies serve as a substrate for the enzyme Dicer in the cytoplasm of acell. In addition to cleaving double-stranded nucleic acid into shortersegments, Dicer may facilitate the incorporation of a single-strandedcleavage product derived from the cleaved dsRNA into the RNA-inducedsilencing complex (RISC complex) that is responsible for the destructionof the cytoplasmic RNA derived from the target gene.

Dicer substrates may have certain properties which enhance itsprocessing by Dicer. Dicer substrates are of a length sufficient suchthat it is processed by Dicer to produce an active nucleic acid moleculeand may further include one or more of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′-overhang on the first strand(antisense strand) and (ii) the dsRNA has a modified 3′-end on thesecond strand (sense strand) to direct orientation of Dicer binding andprocessing of the dsRNA to an active siRNA. In certain embodiments, thelongest strand in the Dicer substrate may be 24-30 nucleotides.

Dicer substrates may be symmetric or asymmetric. The Dicer substrate mayhave a sense strand includes 22-28 nucleotides and the antisense strandmay include 24-30 nucleotides; thus, in some embodiments the resultingDicer substrate may have an overhang on the 3′ end of the antisensestrand. Dicer substrate may have a sense strand 25 nucleotides inlength, and the antisense strand having 27 nucleotides in length with atwo base 3′-overhang. The overhang may be 1-3 nucleotides, for example 2nucleotides. The sense strand may also have a 5′-phosphate.

An asymmetric Dicer substrate may further contain two deoxynucleotidesat the 3′-end of the sense strand in place of two of theribonucleotides. Some exemplary Dicer substrates lengths and structuresare 21+0, 21+2, 21−2, 22+0, 22+1, 22−1, 23+0, 23+2, 23−2, 24+0, 24+2,24−2, 25+0, 25+2, 25−2, 26+0, 26+2, 26−2, 27+0, 27+2, and 27−2.

The sense strand of a Dicer substrate may be between about 22 to about30 (e.g., about 22, 23, 24, 25, 26, 27, 28, 29 or 30); about 22 to about28; about 24 to about 30; about 25 to about 30; about 26 to about 30;about 26 and 29; or about 27 to about 28 nucleotides in length. Incertain preferred embodiments Dicer substrates contain sense andantisense strands, that are at least about 25 nucleotides in length andno longer than about 30 nucleotides; between about 26 and 29nucleotides; or 27 nucleotides in length. The sense and antisensestrands may be the same length (blunt ended), different lengths (haveoverhangs), or a combination. The sense and antisense strands may existon the same polynucleotide or on different polynucleotides. A Dicersubstrate may have a duplex region of about 19, 20, 21, 22, 23, 24, 25or 27 nucleotides.

Like other siNA molecules provided herein, the antisense strand of aDicer substrate may have any sequence that anneals to the antisensestrand under biological conditions, such as within the cytoplasm of aeukaryotic cell.

Dicer substrates may have any modifications to the nucleotide base,sugar or phosphate backbone as known in the art and/or as describedherein for other nucleic acid molecules (such as siNA molecules). Incertain embodiments, Dicer substrates may have a sense strand modifiedfor Dicer processing by suitable modifiers located at the 3′-end of thesense strand, i.e., the dsRNA is designed to direct orientation of Dicerbinding and processing. Suitable modifiers include nucleotides such asdeoxyribonucleotides, dideoxyribonucleotides, acyclo-nucleotides and thelike and sterically hindered molecules, such as fluorescent moleculesand the like. Acyclo-nucleotides substitute a 2-hydroxyethoxymethylgroup for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs.Other nucleotides modifiers that could be used in Dicer substrate siNAmolecules include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, they may replace ribonucleotides (e.g., 1-3 nucleotidemodifiers, or 2 nucleotide modifiers are substituted for theribonucleotides on the 3′-end of the sense strand) such that the lengthof the Dicer substrate does not change. When sterically hinderedmolecules are utilized, they may be attached to the ribonucleotide atthe 3′-end of the antisense strand. Thus, in certain embodiments, thelength of the strand does not change with the incorporation of themodifiers. In certain embodiments, two DNA bases in the dsRNA aresubstituted to direct the orientation of Dicer processing of theantisense strand. In a further embodiment, two terminal DNA bases aresubstituted for two ribonucleotides on the 3′-end of the sense strandforming a blunt end of the duplex on the 3′-end of the sense strand andthe 5′-end of the antisense strand, and a two-nucleotide RNA overhang islocated on the 3′-end of the antisense strand. This is an asymmetriccomposition with DNA on the blunt end and RNA bases on the overhangingend.

In certain embodiments, modifications are included in the Dicersubstrate such that the modification does not prevent the nucleic acidmolecule from serving as a substrate for Dicer. In one embodiment, oneor more modifications are made that enhance Dicer processing of theDicer substrate. One or more modifications may be made that result inmore effective RNAi generation. One or more modifications may be madethat support a greater RNAi effect. One or more modifications are madethat result in greater potency per each Dicer substrate to be deliveredto the cell. Modifications may be incorporated in the 3′-terminalregion, the 5′-terminal region, in both the 3′-terminal and 5′-terminalregion or at various positions within the sequence. Any number andcombination of modifications can be incorporated into the Dicersubstrate so long as the modification does not prevent the nucleic acidmolecule from serving as a substrate for Dicer. Where multiplemodifications are present, they may be the same or different.Modifications to bases, sugar moieties, the phosphate backbone, andtheir combinations are contemplated. Either 5′-terminus can bephosphorylated.

Examples of Dicer substrate phosphate backbone modifications includephosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, and thelike. Examples of Dicer substrate sugar moiety modifications include2′-alkyl pyrimidine, such as 2′OMe, 2′-fluoro, amino, and deoxymodifications and the like (see, e.g., Amarzguioui et al., 2003).Examples of Dicer substrate base group modifications include abasicsugars, 2′-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil,5-iodouracil, and 5-(3-aminoallyl)-uracil and the like. LNAs could alsobe incorporated.

The sense strand may be modified for Dicer processing by suitablemodifiers located at the 3′-end of the sense strand, i.e., the Dicersubstrate is designed to direct orientation of Dicer binding andprocessing. Suitable modifiers include nucleotides such asdeoxyribonucleotides, dideoxyribonucleotides, acyclo-nucleotides and thelike and sterically hindered molecules, such as fluorescent moleculesand the like. Acyclo-nucleotides substitute a 2-hydroxyethoxymethylgroup for-the 2′-deoxyribofuranosyl sugar normally present in dNMPs.Other nucleotides modifiers could include cordycepin, AZT, ddI, 3TC, d4Tand the monophosphate nucleotides of AZT, 3TC and d4T. In oneembodiment, deoxynucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′-end of thesense strand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′-end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the description contemplatessubstituting two DNA bases in the Dicer substrate to direct theorientation of Dicer processing of the antisense strand. In a furtherembodiment of the present description, two terminal DNA bases aresubstituted for two ribonucleotides on the 3′-end of the sense strandforming a blunt end of the duplex on the 3′-end of the sense strand andthe 5′-end of the antisense strand, and a two-nucleotide RNA overhang islocated on the 3′-end of the antisense strand. This is an asymmetriccomposition with DNA on the blunt end and RNA bases on the overhangingend.

The antisense strand may be modified for Dicer processing by suitablemodifiers located at the 3′-end of the antisense strand, i.e., the dsRNAis designed to direct orientation of Dicer binding and processing.Suitable modifiers include nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclo-nucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclo-nucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidesmodifiers could include cordycepin, AZT, ddI, 3TC, d4T and themonophosphate nucleotides of AZT, 3TC and d4T. In one embodiment,deoxynucleotides are used as the modifiers. When nucleotide modifiersare utilized, 1-3 nucleotide modifiers, or 2 nucleotide modifiers aresubstituted for the ribonucleotides on the 3′-end of the antisensestrand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′-end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the description contemplatessubstituting two DNA bases in the dsRNA to direct the orientation ofDicer processing. In a further description, two terminal DNA bases arelocated on the 3′-end of the antisense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′-end of thesense strand and the 3′-end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

Dicer substrates with a sense and an antisense strand can be linked by athird structure. The third structure will not block Dicer activity onthe Dicer substrate and will not interfere with the directed destructionof the RNA transcribed from the target gene. The third structure may bea chemical linking group. Suitable chemical linking groups are known inthe art and can be used. Alternatively, the third structure may be anoligonucleotide that links the two oligonucleotides of the dsRNA is amanner such that a hairpin structure is produced upon annealing of thetwo oligonucleotides making up the Dicer substrate. The hairpinstructure preferably does not block Dicer activity on the Dicersubstrate or interfere with the directed destruction of the RNAtranscribed from the target gene.

The sense and antisense strands of the Dicer substrate are not requiredto be completely complementary. They only need to be substantiallycomplementary to anneal under biological conditions and to provide asubstrate for Dicer that produces a siRNA sufficiently complementary tothe target sequence.

Dicer substrate can have certain properties that enhance its processingby Dicer. The Dicer substrate can have a length sufficient such that itis processed by Dicer to produce an active nucleic acid molecules (e.g.,siRNA) and may have one or more of the following properties: the Dicersubstrate is asymmetric, e.g., has a 3′-overhang on the first strand(antisense strand) and/or the Dicer substrate has a modified 3′ end onthe second strand (sense strand) to direct orientation of Dicer bindingand processing of the Dicer substrate to an active siRNA. The Dicersubstrate can be asymmetric such that the sense strand includes 22-28nucleotides and the antisense strand includes 24-30 nucleotides. Thus,the resulting Dicer substrate has an overhang on the 3′ end of theantisense strand. The overhang is 1-3 nucleotides, for example twonucleotides. The sense strand may also have a 5′ phosphate.

A Dicer substrate may have an overhang on the 3′-end of the antisensestrand and the sense strand is modified for Dicer processing. The 5′-endof the sense strand may have a phosphate. The sense and antisensestrands may anneal under biological conditions, such as the conditionsfound in the cytoplasm of a cell. A region of one of the strands,particularly the antisense strand, of the Dicer substrate may have asequence length of at least 19 nucleotides, wherein these nucleotidesare in the 21-nucleotide region adjacent to the 3′-end of the antisensestrand and are sufficiently complementary to a nucleotide sequence ofthe RNA produced from the target gene. A Dicer substrate may also haveone or more of the following additional properties: the antisense strandhas a right shift from a corresponding 21-mer (i.e., the antisensestrand includes nucleotides on the right side of the molecule whencompared to the corresponding 21-mer), the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairingsand base modifications such as LNA may be included in the 5′-end of thesense strand.

An antisense strand of a Dicer substrate nucleic acid molecule may bemodified to include 1-9 ribonucleotides on the 5′-end to give a lengthof 22-28 nucleotides. When the antisense strand has a length of 21nucleotides, then 1-7 ribonucleotides, or 2-5 ribonucleotides and or 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense strands is not required. That is,the resultant antisense strand is sufficiently complementary with thetarget sequence. A sense strand may then have 24-30 nucleotides. Thesense strand may be substantially complementary with the antisensestrand to anneal to the antisense strand under biological conditions. Inone embodiment, the antisense strand may be synthesized to contain amodified 3′-end to direct Dicer processing. The sense strand may have a3′ overhang. The antisense strand may be synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strandhas a 3′ overhang.

Heat Shock Protein 47

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperoneand resides in the endoplasmic reticulum. It interacts with procollagenduring the process of folding, assembling and transporting from theendoplasmic reticulum (Nagata Trends Biochem Sci 1996; 21:22-6; Razzaqueet al. 2005; Contrib Nephrol 2005; 148: 57-69; Koide et al. 2006 J.Biol. Chem.; 281: 3432-38; Leivo et al. Dev. Biol. 1980; 76:100-114;Masuda et al. J. Clin. Invest. 1994; 94:2481-2488; Masuda et al. CellStress Chaperones 1998; 3:256-264). HSP47 has been reported to have anupregulated expression in various tissue fibrosis (Koide et al. J BiolChem 1999; 274: 34523-26), such as liver cirrhosis (Masuda et al. J ClinInvest 1994; 94:2481-8), pulmonary fibrosis (Razzaque et al. VirchowsArch 1998; 432:455-60; Kakugawa et al. Eur Respir J 2004; 24: 57-65),and glomerulosclerosis (Moriyama et al. Kidney Int 1998; 54: 110-19).Exemplary nucleic acid sequence of target human hsp47 cDNA is disclosedin GenBank accession number: NM 001235 and the corresponding mRNAsequence, for example as listed as SEQ ID NO:1. One of ordinary skill inthe art would understand that a given sequence may change over time andto incorporate any changes needed in the nucleic acid molecules hereinaccordingly.

The specific association of HSP47 with a diverse range of collagen typesmakes HSP47 a potential target for the treatment of fibrosis. Inhibitionof hsp47 expression may prevent extracellular collagen I secretion. Satoet al. (Nat Biotechnol 2008; 26:431-442) explored this possibility byusing siRNA for the inhibition hsp47 expression and preventing theprogression of hepatic fibrosis in rats. Similarly, Chen et al. (Br JDermatol 2007; 156: 1188-1195) and Wang et al. (Plast. Reconstr Surg2003; 111: 1980-7) investigated the inhibition hsp47 expression by RNAinterference technology.

Methods and Compositions for Inhibiting hsp47

Provided are compositions and methods for inhibition of hsp47 expressionby using small nucleic acid molecules, such as short interfering nucleicacid (siNA), interfering RNA (RNAi), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules capable of mediating or that mediate RNA interferenceagainst hsp47 gene expression. The composition and methods disclosedherein are also useful in treating various fibrosis such as liverfibrosis, lung fibrosis, and kidney fibrosis.

Nucleic acid molecule(s) and/or methods of the description are used todown regulate the expression of gene(s) that encode RNA referred to, byexample, Genbank Accession NM_001235.

Compositions, methods and kits provided herein may include one or morenucleic acid molecules (e.g., siNA) and methods that independently or incombination modulate (e.g., downregulate) the expression of hsp47protein and/or genes encoding hsp47 proteins, proteins and/or genesencoding hsp47 (e.g., genes encoding sequences comprising thosesequences referred to by GenBank Accession Nos. NM_001235), or an hsp47gene family member where the genes or gene family sequences sharesequence homology associated with the maintenance and/or development ofdiseases, conditions or disorders associated with hsp47, such as liverfibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritonealfibrosis, chronic hepatic damage, and fibrillogenesis. The descriptionof the various aspects and embodiments is provided with reference toexemplary gene hsp47. However, the various aspects and embodiments arealso directed to other related hsp47 genes, such as homolog genes andtranscript variants, and polymorphisms (e.g., single nucleotidepolymorphism, (SNPs)) associated with certain hsp47 genes. As such, thevarious aspects and embodiments are also directed to other genes thatare involved in hsp47 mediated pathways of signal transduction or geneexpression that are involved, for example, in the maintenance ordevelopment of diseases, traits, or conditions described herein. Theseadditional genes can be analyzed for target sites using the methodsdescribed for the hsp47 gene herein. Thus, the modulation of other genesand the effects of such modulation of the other genes can be performed,determined, and measured as described herein.

In one embodiment, compositions and methods provided herein include adouble-stranded short interfering nucleic acid (siNA) molecule thatdown-regulates expression of a hsp47 gene (e.g., human hsp47 exemplifiedby SEQ ID NO:1), where the nucleic acid molecule includes about 15 toabout 49 base pairs.

In one embodiment, a nucleic acid disclosed may be used to inhibit theexpression of the hsp47 gene or an hsp47 gene family where the genes orgene family sequences share sequence homology. Such homologous sequencescan be identified as is known in the art, for example using sequencealignments. Nucleic acid molecules can be designed to target suchhomologous sequences, for example using perfectly complementarysequences or by incorporating non-canonical base pairs, for examplemismatches and/or wobble base pairs that can provide additional targetsequences. In instances where mismatches are identified, non-canonicalbase pairs (for example, mismatches and/or wobble bases) can be used togenerate nucleic acid molecules that target more than one gene sequence.In a non-limiting example, non-canonical base pairs such as UU and CCbase pairs are used to generate nucleic acid molecules that are capableof targeting sequences for differing hsp47 targets that share sequencehomology. As such, one advantage of using siNAs disclosed herein is thata single nucleic acid can be designed to include nucleic acid sequencethat is complementary to the nucleotide sequence that is conservedbetween the homologous genes. In this approach, a single nucleic acidcan be used to inhibit expression of more than one gene instead of usingmore than one nucleic acid molecule to target the different genes.

Nucleic acid molecules may be used to target conserved sequencescorresponding to a gene family or gene families such as hsp47 familygenes. As such, nucleic acid molecules targeting multiple hsp47 targetscan provide increased therapeutic effect. In addition, nucleic acid canbe used to characterize pathways of gene function in a variety ofapplications. For example, nucleic acid molecules can be used to inhibitthe activity of target gene(s) in a pathway to determine the function ofuncharacterized gene(s) in gene function analysis, mRNA functionanalysis, or translational analysis. The nucleic acid molecules can beused to determine potential target gene pathways involved in variousdiseases and conditions toward pharmaceutical development. The nucleicacid molecules can be used to understand pathways of gene expressioninvolved in, for example fibroses such as liver, kidney or pulmonaryfibrosis, and/or inflammatory and proliferative traits, diseases,disorders, and/or conditions.

In one embodiment, the compositions and methods provided herein includea nucleic acid molecule having RNAi activity against hsp47 RNA, wherethe nucleic acid molecule includes a sequence complementary to any RNAhaving hsp47 encoding sequence, such as those sequences having sequencesas shown in Table 3. In another embodiment, a nucleic acid molecule mayhave RNAi activity against hsp47 RNA, where the nucleic acid moleculeincludes a sequence complementary to an RNA having variant hsp47encoding sequence, for example other mutant hsp47 genes not shown inTable 3 but known in the art to be associated with the maintenanceand/or development of fibrosis. Chemical modifications as shown in Table3 or otherwise described herein can be applied to any nucleic acidconstruct disclosed herein. In another embodiment, a nucleic acidmolecule disclosed herein includes a nucleotide sequence that caninteract with nucleotide sequence of a hsp47 gene and thereby mediatesilencing of hsp47 gene expression, for example, wherein the nucleicacid molecule mediates regulation of hsp47 gene expression by cellularprocesses that modulate the chromatin structure or methylation patternsof the hsp47 gene and prevent transcription of the hsp47 gene.

Nucleic acid molecules disclosed herein may have RNAi activity againsthsp47 RNA, where the nucleic acid molecule includes a sequencecomplementary to any RNA having hsp47 encoding sequence, such as thosesequences having GenBank Accession Nos. NM_001235. Nucleic acidmolecules may have RNAi activity against hsp47 RNA, where the nucleicacid molecule includes a sequence complementary to an RNA having varianthsp47 encoding sequence, for example other mutant hsp47 genes known inthe art to be associated with the maintenance and/or development offibrosis.

Nucleic acid molecules disclosed herein include a nucleotide sequencethat can interact with nucleotide sequence of a hsp47 gene and therebymediate silencing of hsp47 gene expression, e.g., where the nucleic acidmolecule mediates regulation of hsp47 gene expression by cellularprocesses that modulate the chromatin structure or methylation patternsof the hsp47 gene and prevent transcription of the hsp47 gene.

Methods of Treatment

The specific association of HSP47 with a diverse range of collagen typesmakes hsp47 a target for the treatment of fibrosis. Inhibition of hsp47expression may prevent extracellular collagen I secretion. Sato et al.(Nat Biotechnol 2008; 26:431-442) explored this possibility by usingsiRNA for the inhibition hsp47 expression and preventing the progressionof hepatic fibrosis in rats. Similarly, Chen et al. (Br J Dermatol 2007;156: 1188-1195) and Wang et al. (Plast Reconstr Surg 2003; 111: 1980-7)investigated the inhibition hsp47 expression by RNA interferencetechnology.

In one embodiment, nucleic acid molecules may be used to down regulateor inhibit the expression of hsp47 and/or hsp47 proteins arising fromhsp47 and/or hsp47 haplotype polymorphisms that are associated with adisease or condition, (e.g., fibrosis). Analysis of hsp47 and/or hsp47genes, or hsp47 and/or hsp47 protein or RNA levels can be used toidentify subjects with such polymorphisms or those subjects who are atrisk of developing traits, conditions, or diseases described herein.These subjects are amenable to treatment, for example, treatment withnucleic acid molecules disclosed herein and any other composition usefulin treating diseases related to hsp47 and/or hsp47 gene expression. Assuch, analysis of hsp47 and/or hsp47 protein or RNA levels can be usedto determine treatment type and the course of therapy in treating asubject. Monitoring of hsp47 and/or hsp47 protein or RNA levels can beused to predict treatment outcome and to determine the efficacy ofcompounds and compositions that modulate the level and/or activity ofcertain hsp47 and/or hsp47 proteins associated with a trait, condition,or disease.

Provided are compositions and methods for inhibition of hsp47 expressionby using small nucleic acid molecules as provided herein, such as siNA,RNAi, siRNA, double-stranded RNA (dsRNA), micro-RNA (miRNA), and shorthairpin RNA (shRNA) molecules capable of mediating or that mediate RNAinterference against hsp47 gene expression. The composition and methodsdisclosed herein are also useful in treating various fibrosis such asliver fibrosis, lung fibrosis, and kidney fibrosis.

The nucleic acid molecules disclosed herein individually, or incombination or in conjunction with other drugs, can be used forpreventing or treating diseases, traits, conditions and/or disordersassociated with hsp47, such as liver fibrosis, cirrhosis, pulmonaryfibrosis, kidney fibrosis, peritoneal fibrosis, chronic hepatic damage,and fibrillogenesis.

The nucleic acid molecules disclosed herein are able to inhibit theexpression of hsp47 in a sequence specific manner. The nucleic acidmolecules may include a sense strand and an antisense strand whichinclude contiguous nucleotides that are at least partially complementary(antisense) to an hsp47 mRNA.

In some embodiments, dsRNA specific for hsp47 can be used in conjunctionwith other dsRNA specific for other molecular chaperones that assist inthe folding of newly synthesized proteins such as, calnexin,calreticulin, and/or BiP (Bergeron et al. Trends Biochem. Sci. 1994;19:124-128; Herbert et al. 1995; Cold Spring Harb. Symp. Quant. Biol.60:405-415)

Fibrosis can be treated by RNA interference using nucleic acid moleculesas disclosed herein. Exemplary fibroses include liver fibrosis,peritoneal fibrosis, lung fibrosis, kidney fibrosis. The nucleic acidmolecules disclosed herein may inhibit the expression of hsp47 in asequence specific manner.

Treatment of fibrosis can be monitored by determining the level ofextracellular collagen using suitable techniques known in the art suchas, using anti-collagen I antibodies. Treatment can also be monitored bydetermining the level of hsp47 mRNA or the level of HSP47 protein in thecells of the affected tissue. Treatment can also be monitored bynon-invasive scanning of the affected organ or tissue such as bycomputer assisted tomography scan, magnetic resonance elastographyscans.

A method for treating or preventing hsp47 associated disease orcondition in a subject or organism may include contacting the subject ororganism with a nucleic acid molecule as provided herein underconditions suitable to modulate the expression of the hsp47 gene in thesubject or organism.

A method for treating or preventing fibrosis in a subject or organismmay include contacting the subject or organism with a nucleic acidmolecule under conditions suitable to modulate the expression of thehsp47 gene in the subject or organism.

A method for treating or preventing one or more fibroses selected fromthe group consisting of liver fibrosis, kidney fibrosis, and pulmonaryfibrosis in a subject or organism may include contacting the subject ororganism with a nucleic acid molecule under conditions suitable tomodulate the expression of the hsp47 gene in the subject or organism.

Fibrotic Diseases

Fibrotic diseases are generally characterized by the excess depositionof a fibrous material within the extracellular matrix, which contributesto abnormal changes in tissue architecture and interferes with normalorgan function.

All tissues damaged by trauma respond by the initiation of awound-healing program. Fibrosis, a type of disorder characterized byexcessive scarring, occurs when the normal self-limiting process ofwound healing response is disturbed, and causes excessive production anddeposition of collagen. As a result, normal organ tissue is replacedwith scar tissue, which eventually leads to the functional failure ofthe organ.

Fibrosis may be initiated by diverse causes and in various organs. Livercirrhosis, pulmonary fibrosis, sarcoidosis, keloids and kidney fibrosisare all chronic conditions associated with progressive fibrosis, therebycausing a continuous loss of normal tissue function.

Acute fibrosis (usually with a sudden and severe onset and of shortduration) occurs as a common response to various forms of traumaincluding accidental injuries (particularly injuries to the spine andcentral nervous system), infections, surgery, ischemic illness (e.g.cardiac scarring following heart attack), burns, environmentalpollutants, alcohol and other types of toxins, acute respiratorydistress syndrome, radiation and chemotherapy treatment.

Fibrosis, a fibrosis related pathology or a pathology related toaberrant crosslinking of cellular proteins may all be treated by thesiRNAs disclosed herein. Fibrotic diseases or diseases in which fibrosisis evident (fibrosis related pathology) include both acute and chronicforms of fibrosis of organs, including all etiological variants of thefollowing: pulmonary fibrosis, including interstitial lung disease andfibrotic lung disease, liver fibrosis, cardiac fibrosis includingmyocardial fibrosis, kidney fibrosis including chronic renal failure,skin fibrosis including scleroderma, keloids and hypertrophic scars;myelofibrosis (bone marrow fibrosis); all types of ocular scarringincluding proliferative vitreoretinopathy (PVR) and scarring resultingfrom surgery to treat cataract or glaucoma; inflammatory bowel diseaseof variable etiology, macular degeneration, Grave's ophthalmopathy, druginduced ergotism, keloid scars, scleroderma, psoriasis, glioblastoma inLi-Fraumeni syndrome, sporadic glioblastoma, myleoid leukemia, acutemyelogenous leukemia, myelodysplastic syndrome, myeloproferativesyndrome, gynecological cancer, Kaposi's sarcoma, Hansen's disease, andcollagenous colitis.

In various embodiments, the compounds (nucleic acid molecules) asdisclosed herein may be used to treat fibrotic diseases, for example asdisclosed herein, as well as many other diseases and conditions apartfrom fibrotic diseases, for example such as disclosed herein. Otherconditions to be treated include fibrotic diseases in otherorgans—kidney fibrosis for any reason (CKD including ESRD); lungfibrosis (including ILF); myelofibrosis, abnormal scarring (keloids)associated with all possible types of skin injury accidental andjatrogenic (operations); scleroderma; cardiofibrosis, failure ofglaucoma filtering operation; intestinal adhesions.

Ocular Surgery and Fibrotic Complications

Contracture of scar tissue resulting from eye surgery may often occur.Glaucoma surgery to create new drainage channels often fails due toscarring and contraction of tissues and the generated drainage systemmay be blocked requiring additional surgical intervention. Currentanti-scarring regimens (Mitomycin C or 5FU) are limited due to thecomplications involved (e.g. blindness) e.g. see Cordeiro M F, et al.,Human anti-transforming growth factor-beta2 antibody: a new glaucomaanti-scarring agent Invest Ophthalmol Vis Sci. 1999 September;40(10):2225-34. There may also be contraction of scar tissue formedafter corneal trauma or corneal surgery, for example laser or surgicaltreatment for myopia or refractive error in which contraction of tissuesmay lead to inaccurate results. Scar tissue may be formed on/in thevitreous humor or the retina, for example, and may eventually causesblindness in some diabetics, and may be formed after detachment surgery,called proliferative vitreoretinopathy (PVR). PVR is the most commoncomplication following retinal detachment and is associated with aretinal hole or break. PVR refers to the growth of cellular membraneswithin the vitreous cavity and on the front and back surfaces of theretina containing retinal pigment epithelial (RPE) cells. Thesemembranes, which are essentially scar tissues, exert traction on theretina and may result in recurrences of retinal detachment, even afteran initially successful retinal detachment procedure.

Scar tissue may be formed in the orbit or on eye and eyelid musclesafter squint, orbital or eyelid surgery, or thyroid eye disease, andwhere scarring of the conjunctiva occurs as may happen after glaucomasurgery or in cicatricial disease, inflammatory disease, for example,pemphigoid, or infective disease, for example, trachoma. A further eyeproblem associated with the contraction of collagen-including tissues isthe opacification and contracture of the lens capsule after cataractextraction. Important role for MMPs has been recognized in oculardiseases including wound healing, dry eye, sterile corneal ulceration,recurrent epithelial erosion, corneal neovascularization, pterygium,conjunctivochalasis, glaucoma, PVR, and ocular fibrosis.

Liver Fibrosis

Liver fibrosis (LF) is a generally irreversible consequence of hepaticdamage of several etiologies. In the Western world, the main etiologiccategories are: alcoholic liver disease (30-50%), viral hepatitis (30%),biliary disease (5-10%), primary hemochromatosis (5%), and drug-relatedand cryptogenic cirrhosis of, unknown etiology, (10-15%). Wilson'sdisease, α₁-antitrypsin deficiency and other rare diseases also haveliver fibrosis as one of the symptoms. Liver cirrhosis, the end stage ofliver fibrosis, frequently requires liver transplantation and is amongthe top ten causes of death in the Western world.

Kidney Fibrosis and Related Conditions

Chronic Renal Failure (CRF)

Chronic renal failure is a gradual and progressive loss of the abilityof the kidneys to excrete wastes, concentrate urine, and conserveelectrolytes. CRF is slowly progressive. It most often results from anydisease that causes gradual loss of kidney function, and fibrosis is themain pathology that produces CRF.

Diabetic Nephropathy

Diabetic nephropathy, hallmarks of which are glomerulosclerosis andtubulointerstitial fibrosis, is the single most prevalent cause ofend-stage renal disease in the modern world, and diabetic patientsconstitute the largest population on dialysis. Such therapy is costlyand far from optimal. Transplantation offers a better outcome butsuffers from a severe shortage of donors.

Chronic Kidney Disease

Chronic kidney disease (CKD) is a worldwide public health problem and isrecognized as a common condition that is associated with an increasedrisk of cardiovascular disease and chronic renal failure (CRF).

The Kidney Disease Outcomes Quality Initiative (K/DOQI) of the NationalKidney Foundation (NKF) defines chronic kidney disease as either kidneydamage or a decreased kidney glomerular filtration rate (GFR) for threeor more months. Other markers of CKD are also known and used fordiagnosis. In general, the destruction of renal mass with irreversiblesclerosis and loss of nephrons leads to a progressive decline in GFR.Recently, the K/DOQI published a classification of the stages of CKD, asfollows:

Stage 1: Kidney damage with normal or increased GFR (>90 mL/min/1.73 m2)

Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m2)

Stage 3: Moderate reduction in GFR (30-59 mL/min/1.73 m2)

Stage 4: Severe reduction in GFR (15-29 mL/min/1.73 m2)

Stage 5: Kidney failure (GFR <15 mL/min/1.73 m2 or dialysis)

In stages 1 and 2 CKD, GFR alone does not confirm the diagnosis. Othermarkers of kidney damage, including abnormalities in the composition ofblood or urine or abnormalities in imaging tests, may be relied upon.

Pathophysiology of CKD

Approximately 1 million nephrons are present in each kidney, eachcontributing to the total GFR. Irrespective of the etiology of renalinjury, with progressive destruction of nephrons, the kidney is able tomaintain GFR by hyperfiltration and compensatory hypertrophy of theremaining healthy nephrons. This nephron adaptability allows forcontinued normal clearance of plasma solutes so that substances such asurea and creatinine start to show significant increases in plasma levelsonly after total GFR has decreased to 50%, when the renal reserve hasbeen exhausted. The plasma creatinine value will approximately doublewith a 50% reduction in GFR. Therefore, a doubling in plasma creatininefrom a baseline value of 0.6 mg/dL to 1.2 mg/dL in a patient actuallyrepresents a loss of 50% of functioning nephron mass.

The residual nephron hyperfiltration and hypertrophy, althoughbeneficial for the reasons noted, is thought to represent a major causeof progressive renal dysfunction. This is believed to occur because ofincreased glomerular capillary pressure, which damages the capillariesand leads initially to focal and segmental glomerulosclerosis andeventually to global glomerulosclerosis. This hypothesis has been basedon studies of five-sixths nephrectomized rats, which develop lesionsthat are identical to those observed in humans with CKD.

The two most common causes of chronic kidney disease are diabetes andhypertension. Other factors include acute insults from nephrotoxins,including contrasting agents, or decreased perfusion; proteinuria;increased renal ammoniagenesis with interstitial injury; hyperlipidemia;Hyperphosphatemia with calcium phosphate deposition; decreased levels ofnitrous oxide and smoking.

In the United States, the incidence and prevalence of CKD is rising,with poor outcomes and high cost to the health system. Kidney disease isthe ninth leading cause of death in the US. The high rate of mortalityhas led the US Surgeon General's mandate for America's citizenry,Healthy People 2010, to contain a chapter focused on CKD. The objectivesof this chapter are to articulate goals and to provide strategies toreduce the incidence, morbidity, mortality, and health costs of chronickidney disease in the United States.

The incidence rates of end-stage renal disease (ESRD) have alsoincreased steadily internationally since 1989. The United States has thehighest incident rate of ESRD, followed by Japan. Japan has the highestprevalence per million population followed by the United States.

The mortality rates associated with hemodialysis are striking andindicate that the life expectancy of patients entering into hemodialysisis markedly shortened. At every age, patients with ESRD on dialysis havesignificantly increased mortality when compared with nondialysispatients and individuals without kidney disease. At age 60 years, ahealthy person can expect to live for more than 20 years, whereas thelife expectancy of a 60-year-old patient starting hemodialysis is closerto 4 years.

Pulmonary Fibrosis

Interstitial pulmonary fibrosis (IPF) is scarring of the lung caused bya variety of inhaled agents including mineral particles, organic dusts,and oxidant gases, or by unknown reasons (idiopathic lung fibrosis). Thedisease afflicts millions of individuals worldwide, and there are noeffective therapeutic approaches. A major reason for the lack of usefultreatments is that few of the molecular mechanisms of disease have beendefined sufficiently to design appropriate targets for therapy (Lasky,et al., 2000, Environ Health Perspect; 108:751-62).

Cardiac Fibrosis

Heart failure is unique among the major cardiovascular disorders in thatit alone is increasing in prevalence while there has been a strikingdecrease in other conditions. Some of this can be attributed to theaging of the populations of the United States and Europe. The ability tosalvage patients with myocardial damage is also a major factor, as thesepatients may develop progression of left ventricular dysfunction due todeleterious remodelling of the heart.

The normal myocardium is composed of a variety of cells, cardiacmyocytes and noncardiomyocytes, which include endothelial and vascularsmooth muscle cells and fibroblasts.

Structural remodeling of the ventricular wall is a key determinant ofclinical outcome in heart disease. Such remodeling involves theproduction and destruction of extracellular matrix proteins, cellproliferation and migration, and apoptotic and necrotic cell death.Cardiac fibroblasts are crucially involved in these processes, producinggrowth factors and cytokines that act as autocrine and paracrinefactors, as well as extracellular matrix proteins and proteinases.Recent studies have shown that the interactions between cardiacfibroblasts and cardiomyocytes are essential for the progression ofcardiac remodeling of which the net effect is deterioration in cardiacfunction and the onset of heart failure (Manabe, et al., 2002, Circ Res.13:1103-13).

Burns and Scars

A particular problem which may arise, particularly in fibrotic disease,is contraction of tissues, for example contraction of scars. Contractionof tissues including extracellular matrix components, especially ofcollagen-including tissues, may occur in connection with many differentpathological conditions and with surgical or cosmetic procedures.Contracture, for example, of scars, may cause physical problems, whichmay lead to the need for medical treatment, or it may cause problems ofa purely cosmetic nature. Collagen is the major component of scar andother contracted tissue and as such is the most important structuralcomponent to consider. Nevertheless, scar and other contracted tissuealso include other structural components, especially other extracellularmatrix components, for example, elastin, which may also contribute tocontraction of the tissue.

Contraction of collagen-including tissue, which may also include otherextracellular matrix components, frequently occurs in the healing ofburns. The burns may be chemical, thermal or radiation burns and may beof the eye, the surface of the skin or the skin and the underlyingtissues. It may also be the case that there are burns on internaltissues, for example, caused by radiation treatment. Contraction ofburnt tissues is often a problem and may lead to physical and/orcosmetic problems, for example, loss of movement and/or disfigurement.

Skin grafts may be applied for a variety of reasons and may oftenundergo contraction after application. As with the healing of burnttissues the contraction may lead to both physical and cosmetic problems.It is a particularly serious problem where many skin grafts are neededas, for example, in a serious burns case.

Contraction is also a problem in production of artificial skin. To makea true artificial skin it is necessary to have an epidermis made ofepithelial cells (keratinocytes) and a dermis made of collagen populatedwith fibroblasts. It is important to have both types of cells becausethey signal and stimulate each other using growth factors. The collagencomponent of the artificial skin often contracts to less than one tenthof its original area when populated by fibroblasts.

Cicatricial contraction, contraction due to shrinkage of the fibroustissue of a scar, is common. In some cases the scar may become a viciouscicatrix, a scar in which the contraction causes serious deformity. Apatient's stomach may be effectively separated into two separatechambers in an hour-glass contracture by the contraction of scar tissueformed when a stomach ulcer heals. Obstruction of passages and ducts,cicatricial stenosis, may occur due to the contraction of scar tissue.Contraction of blood vessels may be due to primary obstruction orsurgical trauma, for example, after surgery or angioplasty. Stenosis ofother hollow viscus, for examples, ureters, may also occur. Problems mayoccur where any form of scarring takes place, whether resulting fromaccidental wounds or from surgery. Conditions of the skin and tendonswhich involve contraction of collagen-including tissues includepost-trauma conditions resulting from surgery or accidents, for example,hand or foot tendon injuries, post-graft conditions and pathologicalconditions, such as scleroderma, Dupuytren's contracture andepidermolysis bullosa. Scarring and contraction of tissues in the eyemay occur in various conditions, for example, the sequelae of retinaldetachment or diabetic eye disease (as mentioned above). Contraction ofthe sockets found in the skull for the eyeballs and associatedstructures, including extra-ocular muscles and eyelids, may occur ifthere is trauma or inflammatory damage. The tissues contract within thesockets causing a variety of problems including double vision and anunsightly appearance.

For further information on different types of fibrosis see: Molina V, etal., 2002, Harefuah, 141: 973-8, 1009; Yu, et al., 2002, Curr OpinPharmacol. 2(2):177-81; Keane, et al., 2003, Am J Kidney Dis. 41: S22-5;Bohle, et al., 1989, Pathol Res Pract. 185:421-40; Kikkawa, et al.,1997, Kidney Int Suppl. 62:S39-40; Bataller et al., 2001, Semin LiverDis. 21:437-51; Gross, et al., 2001 N Engl J Med. 345:517-25; Frohlich,2001, Am J Hypertens; 14:194S-199S; Friedman, 2003, J Hepatol.38:S38-53; Albanis, et al., 2003, Curr Gastroenterol Rep. 5:48-56;Weber, 2000, Curr Opin Cardiol. 15:264-72).

Delivery of Nucleic Acid Molecules and Pharmaceutical Formulations

The retinoid or retinoid conjugate useful for delivery of nucleic acidis in a state in which it is dissolved in or mixed with a medium thatcan dissolve or retain it.

Any retinoid or retinoid conjugate may be used in the presentdescription as long as it is actively accumulated by stellate cells;examples of retinoid include, but are not limited to, tretinoin,adapalene, retinol palmitate, and in particular vitamin A, saturatedvitamin A, retinoic acid, and retinal. Examples of theretinoid-conjugate include PEG-retinoid conjugates. The presentdescription utilizes the property of stellate cells to positivelyincorporate a retinoid and/or a retinoid conjugate, and by using theretinoid and/or retinoid conjugate as a drug carrier or by bonding to orbeing included in another drug carrier component, a desired material orbody is transported specifically to stellate cells. A retinoid is amember of the class of compounds having a skeleton in which fourisoprenoid units are bonded in a head-to-tail manner. See G. P. Moss,“Biochemical Nomenclature and Related Documents,” 2nd Ed. PortlandPress, pp. 247-251 (1992). Vitamin A is a generic descriptor for aretinoid qualitatively showing the biological activity of retinol. Theretinoid in the present description promotes specific substance deliveryto a cancer cell and a CAF (that is, the substance is targeted at thesecells). Such a retinoid is not particularly limited, and examplesthereof include retinol, Vitamin A, saturated Vitamin A, retinal,retinoic acid, an ester of retinol and a fatty acid, an ester of analiphatic alcohol and retinoic acid, etretinate, tretinoin,isotretinoin, adapalene, acitretine, tazarotene, and retinol palmitate,and vitamin A analogues such as fenretinide, and bexarotene.Retinoid-conjugates include PEG-conjugates, e.g., diVA-PEG-diVA, shownin the following structure.

The drug carrier of the present description therefore may contain a drugcarrier component other than a retinoid and/or retinoid-conjugate. Sucha component is not particularly limited, and any component known in thefields of medicine and pharmacy may be used, but it is preferable for itto be capable of including a retinoid and/or retinoid conjugate.Examples of such a component include a lipid, for example, aphospholipid such as glycerophospholipid, a sphingolipid such assphingomyelin, a sterol such as cholesterol, a vegetable oil such assoybean oil or poppy seed oil, mineral oil, and a lecithin such asegg-yolk lecithin, but the examples are not limited thereto. Among them,those that can form a liposome are preferable, for example, naturalphospholipids such as lecithin, semisynthetic phospholipids such asdimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine(DPPC), and distearoylphosphatidylcholine (DSPC), and cholesterol.

Furthermore, the drug carrier of the present description may contain asubstance that improves incorporation into stellate cells, for example,retinol-binding protein (RBP).

The bonding or inclusion of the retinoid and/or retinoid conjugate withthe drug carrier of the present description may also be carried out bybonding or including the retinoid and/or retinoid conjugate with anothercomponent of the drug carrier by chemical and/or physical methods.Alternatively, bonding or inclusion of the retinoid and/or retinoidconjugate with the drug carrier of the present description may also becarried out by mixing the retinoid and/or retinoid conjugate havingformation-affinity and basic components of the drug carrier, into thedrug carrier components during preparation of the drug carrier. Theamount of retinoid and/or retinoid conjugate bonded to or included inthe drug carrier of the present description may be 0.01% to 100% as aratio by weight relative to the drug carrier components, preferably 0.2%to 20%, and more preferably 1% to 5%.

Nucleic acid delivery systems may include, for example, aqueous andnonaqueous gels, creams, multiple emulsions, microemulsions, liposomes,ointments, aqueous and nonaqueous solutions, lotions, aerosols,hydrocarbon bases and powders, and can contain excipients such assolubilizers, permeation enhancers (e.g., fatty acids, fatty acidesters, fatty alcohols and amino acids), and hydrophilic polymers (e.g.,polycarbophil and polyvinylpyrolidone). In one embodiment, thepharmaceutically acceptable carrier is a liposome or a transdermalenhancer. Examples of liposomes which can be used in this descriptioninclude the following:

-   -   CellFectin, a 1:1.5 (M/M) liposome formulation of the cationic        lipid        N,N^(I),N^(II),N^(III)-tetramethyl-N,N^(I),N^(II),N^(III)-tetrapalmityl-spermine        and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL);    -   Cytofectin GSV, a 2:1 (M/M) liposome formulation of a cationic        lipid and DOPE (Glen Research);    -   DOTAP        (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)        (Boehringer Manheim);    -   Lipofectamine, a 3:1 (M/M) liposome formulation of the        polycationic lipid DOSPA, the neutral lipid DOPE (GIBCO BRL) and        Di-Alkylated Amino Acid (DiLA2);    -   Lipotrust, a 4:3:3 (M/M) liposome formulation of        O,O′-ditetradecanoyl-N-(α-trimethylammonioacetyl) diethanolamine        chloride (DC-6-14, cholesterol and        dioleoylphosphatidylethanolamine (Hokkaido System Science).        DC-6-14 consists of the following structure.

Other lipids may be useful: permanent cationic lipids and ionizablecationic lipids, including

and PEG-lipids, including

-   -   1,2-dimyristoleoyl-sn-glycero-3-phosphoethanolamine-N-PEG        (PEG-DMPE)    -   1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-PEG        (PEG-DPPE),    -   1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG        (PEG-DSPE), or    -   1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-PEG (PEG-DOPE)        and/or    -   PEG-ceramide.

Delivery systems may include patches, tablets, suppositories, pessaries,gels and creams, and can contain excipients such as solubilizers andenhancers (e.g., propylene glycol, bile salts and amino acids), andother vehicles (e.g., polyethylene glycol, fatty acid esters andderivatives, and hydrophilic polymers such ashydroxypropylmethylcellulose and hyaluronic acid).

The drug carrier of the present description may be in any form as longas a desired material or body can be transported to target stellatecells, and examples of the form include, but are not limited to, polymermicelle, liposome, emulsion, microsphere, and nanosphere. Furthermore,the drug carrier of the present description may include in its interiorthe substance that is to be transported, be attached to the exterior ofthe substance that is to be transported, or be mixed with the substancethat is to be transported as long as the retinoid and/or retinoidconjugate included therein is at least partially exposed on the exteriorof the preparation before it reaches the stellate cells at the latest.

The drug carrier of the present description specifically targetsstellate cells and enables a desired effect such as, for example,inhibition or prevention of fibrosis to be exhibited with the maximumeffect and minimum side effects by efficiently transporting to stellatecells a desired material or body such as, for example, a drug forcontrolling the activity or growth of stellate cells. The material orbody that the present drug carrier delivers is not particularly limited,but it preferably has a size that enables physical movement in a livingbody from an administration site to the liver, pancreas, etc., wherestellate cells are present. The drug carrier of the present descriptiontherefore can transport not only a material such as an atom, a molecule,a compound, a protein, or a nucleic acid but also a body such as avector, a virus particle, a cell, a drug release system constituted fromone or more elements, or a micromachine. The material or body preferablyhas the property of exerting some effect on stellate cells, and examplesthereof include one that labels stellate cells and one that controls theactivity or growth of stellate cells.

Therefore, in one embodiment of the present description, it is a drugfor controlling the activity or growth of stellate cells that the drugcarrier delivers. This may be any drug that directly or indirectlyinhibits the physicochemical actions of stellate cells involved in thepromotion of fibrosis, and examples thereof include, but are not limitedto, TGFβ activity inhibitors such as a truncated TGFβ type II receptorand a soluble TGFβ type II receptor, growth factor preparations such asHGF and expression vectors therefor, MMP production promoters such as anMMP gene-containing adenovirus vector, TIMP production inhibitors suchas an antisense TIMP nucleic acid, a PPARγ ligand, cell activationinhibitors and/or cell growth inhibitors such as an angiotensin activityinhibitor, a PDGF activity inhibitor, and a sodium channel inhibitor,and also apoptosis inducers such as compound 861 and gliotoxin,adiponectin, and a compound having Rho kinase inhibitory activity suchas (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane.Furthermore, the ‘drug for controlling the activity or growth ofstellate cells’ in the present description may be any drug that directlyor indirectly promotes the physicochemical actions of stellate cellsdirectly or indirectly involved in the inhibition of fibrosis, andexamples thereof include, but are not limited to, a drug for promoting acollagen degradation system, e.g., MMP production promoters such as anMMP expression vector, HGF, and drugs having HGF-like activity such asHGF analogues and expression vectors therefor.

Other examples of the drug for controlling the activity or growth ofstellate cells in the present description include a drug for controllingthe metabolism of an extracellular matrix such as collagen, for example,a substance having an effect in inhibiting the expression of a targetmolecule, such as siRNA, ribozyme, and antisense nucleic acid (includingRNA, DNA, PNA, and a composite thereof), a substance having a dominantnegative effect, and vectors expressing same, that target, for example,an extracellular matrix constituent molecule produced by stellate cellsor target one or more molecules that have the function of producing orsecreting the extracellular matrix constituent molecule.

The present description also relates to a medicine for treating astellate cell-related disorder, the medicine containing the drug carrierand the drug for controlling the activity or growth of stellate cells,and relates to the use of the drug carrier in the production of apharmaceutical composition for treating a stellate cell-relateddisorder. The stellate cell-related disorder referred to here means adisorder in which stellate cells are directly or indirectly involved inthe process of the disorder, that is, the onset, exacerbation,improvement, remission, cure, etc. of the disorder, and examples thereofinclude hepatic disorders such as hepatitis, in particular chronichepatitis, hepatic fibrosis, hepatic cirrhosis, and liver cancer, andpancreatic disorders such as pancreatitis, in particular chronicpancreatitis, pancreatic fibrosis, and pancreatic cancer.

In the medicine of the present description, the drug carrier may includea drug in its interior, be attached to the exterior of a drug-containingsubstance, or be mixed with a drug as long as the retinoid and/orretinoid-conjugate included in the drug carrier is at least partiallyexposed on the exterior of the preparation before it reaches thestellate cells at the latest. Therefore, depending on the route ofadministration or manner in which the drug is released, the medicine maybe covered with an appropriate material, such as, for example, anenteric coating or a material that disintegrates over time, or may beincorporated into an appropriate drug release system.

The present description therefore includes a drug carrier or medicinepreparation kit containing one or more containers containing one or moreof a drug carrier constituent, a retinoid and/or a retinoid conjugate,and/or a drug, and also includes an essential component for the drugcarrier or the medicine provided in the form of such a kit. The kit ofthe present description may contain, in addition to those describedabove, a description, etc. in which a preparation method or anadministration method for the drug carrier and the medicine of thepresent description is described. Furthermore, the kit of the presentdescription may contain all components for completing the drug carrieror the medicine of the present description but need not necessarilycontain all of the components. The kit of the present descriptiontherefore need not contain a reagent or a solvent that is normallyavailable at a place of medical treatment, an experimental facility,etc. such as, for example, sterile water, saline, or a glucose solution.

The present description further relates to a method for treating astellate cell-related disorder, the method including administering aneffective amount of the medicine to a subject in need thereof. Theeffective amount referred to here is an amount that suppresses onset ofthe target disorder, reduces symptoms thereof, or prevents progressionthereof, and is preferably an amount that prevents onset of the targetdisorder or cures the target disorder. It is also preferably an amountthat does not cause an adverse effect that exceeds the benefit fromadministration. Such an amount may be determined as appropriate by an invitro test using cultured cells, etc. or by a test in a model animalsuch as a mouse, a rat, a dog, or a pig, and such test methods are wellknown to a person skilled in the art.

In the method of the present description, the term ‘subject’ means anyliving individual, preferably an animal, more preferably a mammal, andyet more preferably a human individual. In the present description, thesubject may be healthy or affected with some disorder, and in the caseof treatment of a disorder being intended, the subject typically means asubject affected with the disorder or having a risk of being affected.

Furthermore, the term ‘treatment’ includes all types of medicallyacceptable prophylactic and/or therapeutic intervention for the purposeof the cure, temporary remission, prevention, etc. of a disorder. Forexample, when the disorder is hepatic fibrosis, the term ‘treatment’includes medically acceptable intervention for various purposesincluding delaying or halting the progression of fibrosis, regression ordisappearance of lesions, prevention of the onset of fibrosis, orprevention of recurrence.

The present description also relates to a method for delivering a drugto stellate cells using the drug carrier. This method includes, but isnot limited to, a step of supporting a substance to be delivered on thedrug carrier, and a step of administering or adding the drug carriercarrying the substance to be delivered to a stellate cell-containingliving body or medium, such as, for example, a culture medium. Thesesteps may be achieved as appropriate in accordance with any knownmethod, the method described in the present specification, etc. Thisdelivery method may be combined with another delivery method, forexample, another delivery method in which an organ where stellate cellsare present is the target, etc.

Nucleic acid molecules may be adapted for use to prevent or treatfibroses (e.g., liver, kidney, peritoneal, and pulmonary) diseases,traits, conditions and/or disorders, and/or any other trait, disease,disorder or condition that is related to or will respond to the levelsof hsp47 in a cell or tissue, alone or in combination with othertherapies. A nucleic acid molecule may include a delivery vehicle,including liposomes, for administration to a subject, carriers anddiluents and their salts, and/or can be present in pharmaceuticallyacceptable formulations.

The nucleic acid molecules of the description may include sequencesshown in Table 3. Examples of such nucleic acid molecules consistessentially of sequences provided in Table 3.

The nucleic acid molecules may be administered via pulmonary delivery,such as by inhalation of an aerosol or spray dried formulationadministered by an inhalation device or nebulizer, providing rapid localuptake of the nucleic acid molecules into relevant pulmonary tissues.Solid particulate compositions containing respirable dry particles ofmicronized nucleic acid compositions can be prepared by grinding driedor lyophilized nucleic acid compositions, and then passing themicronized composition through, for example, a 400 mesh screen to breakup or separate out large agglomerates. A solid particulate compositioncomprising the nucleic acid compositions of the description canoptionally contain a dispersant which serves to facilitate the formationof an aerosol as well as other therapeutic compounds. A suitabledispersant is lactose, which can be blended with the nucleic acidcompound in any suitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles may include a nucleic acid moleculesdisclosed herein and can be produced by any suitable means, such as witha nebulizer (see e.g., U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers include the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, e.g., sodium chloride or other suitable salts. Optionaladditives include preservatives if the formulation is not preparedsterile, e.g., methyl hydroxybenzoate, anti-oxidants, flavorings,volatile oils, buffering agents and emulsifiers and other formulationsurfactants. The aerosols of solid particles including the activecomposition and surfactant can likewise be produced with any solidparticulate aerosol generator. Aerosol generators for administeringsolid particulate therapeutics to a subject produce particles which arerespirable, as explained above, and generate a volume of aerosolcontaining a predetermined metered dose of a therapeutic composition ata rate suitable for human administration. One illustrative type of solidparticulate aerosol generator is an insufflator. Suitable formulationsfor administration by insufflation include finely comminuted powderswhich can be delivered by means of an insufflator. In the insufflator,the powder, e.g., a metered dose thereof effective to carry out thetreatments described herein, is contained in capsules or cartridges,typically made of gelatin or plastic, which are either pierced or openedin situ and the powder delivered by air drawn through the device uponinhalation or by means of a manually-operated pump. The powder employedin the insufflator consists either solely of the active ingredient or ofa powder blend comprising the active ingredient, a suitable powderdiluent, such as lactose, and an optional surfactant. The activeingredient typically includes from 0.1 to 100 w/w of the formulation. Asecond type of illustrative aerosol generator includes a metered doseinhaler. Metered dose inhalers are pressurized aerosol dispensers,typically containing a suspension or solution formulation of the activeingredient in a liquefied propellant. During use these devices dischargethe formulation through a valve adapted to deliver a metered volume toproduce a fine particle spray containing the active ingredient. Suitablepropellants include certain chlorofluorocarbon compounds, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, e.g., US20040037780,U.S. Pat. No. 6,592,904, U.S. Pat. No. 6,582,728, and U.S. Pat. No.6,565,885. WO08132723 relates to aerosol delivery of oligonucleotides ingeneral, and of siRNA in particular, to the respiratory system.

Nucleic acid molecules may be administered to the central nervous system(CNS) or peripheral nervous system (PNS). Experiments have demonstratedthe efficient in vivo uptake of nucleic acids by neurons. See e.g.,Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8:75; Epa et al.,2000, Antisense Nuc. Acid Drug Dev., 10:469; Broaddus et al., 1998, J.Neurosurg., 88:734; Karle et al., 1997, Eur. J. Pharmocol., 340:153;Bannai et al., 1998, Brain Research, 784:304; Rajakumar et al., 1997,Synapse, 26:199; Wu-pong et al., 1999, BioPharm, 12:32; Bannai et al.,1998, Brain Res. Protoc., 3:83; and Simantov et al., 1996, Neuroscience,74:39. Nucleic acid molecules are therefore amenable to delivery to anduptake by cells in the CNS and/or PNS.

Delivery of nucleic acid molecules to the CNS is provided by a varietyof different strategies. Traditional approaches to CNS delivery that canbe used include, but are not limited to, intrathecal andintracerebroventricular administration, implantation of catheters andpumps, direct injection or perfusion at the site of injury or lesion,injection into the brain arterial system, or by chemical or osmoticopening of the blood-brain barrier. Other approaches can include the useof various transport and carrier systems, for example through the use ofconjugates and biodegradable polymers. Furthermore, gene therapyapproaches, e.g., as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

Nucleic acid molecules may be formulated or complexed withpolyethylenimine (e.g., linear or branched PEI) and/or polyethyleniminederivatives, including for example grafted PEIs such as galactose PEI,cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI(PEG-PEI) derivatives thereof (see for example Ogris et al., 2001, AAPAPharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14,840-847; Kunath et al., 2002, Pharm Res 19:810-17; Choi et al., 2001,Bull. Korean Chem. Soc., 22:46-52; Bettinger et al., 1999, BioconjugateChem., 10:558-561; Peterson et al., 2002, Bioconjugate Chem. 13:845-54;Erbacher et al., 1999, J Gene Med 1:1-18; Godbey et al., 1999., PNAS,96:5177-81; Godbey et al., 1999, J Controlled Release, 60:149-60;Diebold et al., 1999, J Biol Chem, 274:19087-94; Thomas et al., 2002,PNAS, 99, 14640-45; and Sagara, U.S. Pat. No. 6,586,524).

Nucleic acid molecules may include a bioconjugate, for example a nucleicacid conjugate as described in Vargeese et al., U.S. Ser. No.10/427,160; U.S. Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat.No. 6,235,886; U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S.Pat. No. 5,138,045.

Compositions, methods and kits disclosed herein may include anexpression vector that includes a nucleic acid sequence encoding atleast one nucleic acid molecule of the description in a manner thatallows expression of the nucleic acid molecule. Methods of introducingnucleic acid molecules or one or more vectors capable of expressing thestrands of dsRNA into the environment of the cell will depend on thetype of cell and the make up of its environment. The nucleic acidmolecule or the vector construct may be directly introduced into thecell (i.e., intracellularly); or introduced extracellularly into acavity, interstitial space, into the circulation of an organism,introduced orally, or may be introduced by bathing an organism or a cellin a solution containing dsRNA. The cell is preferably a mammalian cell;more preferably a human cell. The nucleic acid molecule of theexpression vector can include a sense region and an antisense region.The antisense region can include a sequence complementary to a RNA orDNA sequence encoding hsp47 and the sense region can include a sequencecomplementary to the antisense region. The nucleic acid molecule caninclude two distinct strands having complementary sense and antisenseregions. The nucleic acid molecule can include a single-strand havingcomplementary sense and antisense regions.

Nucleic acid molecules that interact with target RNA molecules anddown-regulate gene encoding target RNA molecules (e.g., target RNAmolecules referred to by Genbank Accession numbers herein) may beexpressed from transcription units inserted into DNA or RNA vectors.Recombinant vectors can be DNA plasmids or viral vectors. Nucleic acidmolecule expressing viral vectors can be constructed based on, but notlimited to, adeno-associated virus, retrovirus, adenovirus, oralphavirus. The recombinant vectors capable of expressing the nucleicacid molecules can be delivered as described herein, and persist intarget cells. Alternatively, viral vectors can be used that provide fortransient expression of nucleic acid molecules. Such vectors can berepeatedly administered as necessary. Once expressed, the nucleic acidmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of nucleic acid molecule expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from asubject followed by reintroduction into the subject, or by any othermeans that would allow for introduction into the desired target cell.

Expression vectors may include a nucleic acid sequence encoding at leastone nucleic acid molecule disclosed herein, in a manner which allowsexpression of the nucleic acid molecule. For example, the vector maycontain sequence(s) encoding both strands of a nucleic acid moleculethat include a duplex. The vector can also contain sequence(s) encodinga single nucleic acid molecule that is self-complementary and thus formsa nucleic acid molecule. Non-limiting examples of such expressionvectors are described in Paul et al., 2002, Nature Biotech 19, 505;Miyagishi et al., 2002, Nature Biotech 19, 497; Lee et al., 2002, NatureBiotech 19, 500; and Novina et al., 2002, Nature Med:10.1038/nm725.Expression vectors may also be included in a mammalian (e.g., human)cell.

An expression vector may include a nucleic acid sequence encoding two ormore nucleic acid molecules, which can be the same or different.Expression vectors may include a sequence for a nucleic acid moleculecomplementary to a nucleic acid molecule referred to by a GenbankAccession number NM_001235, for example those shown in Table 2.

An expression vector may encode one or both strands of a nucleic acidduplex, or a single self-complementary strand that self hybridizes intoa nucleic acid duplex. The nucleic acid sequences encoding nucleic acidmolecules can be operably linked in a manner that allows expression ofthe nucleic acid molecule (see for example Paul et al., 2002, NatureBiotech, 19:505; Miyagishi and Taira, 2002, Nature Biotech 19:497; Leeet al., 2002, Nature Biotech 19:500; and Novina et al., 2002, NatureMed, 10.1038/nm725).

An expression vector may include one or more of the following: a) atranscription initiation region (e.g., eukaryotic pol I, II or IIIinitiation region); b) a transcription termination region (e.g.,eukaryotic pol I, II or III termination region); c) an intron and d) anucleic acid sequence encoding at least one of the nucleic acidmolecules, wherein said sequence is operably linked to the initiationregion and the termination region in a manner that allows expressionand/or delivery of the nucleic acid molecule. The vector can optionallyinclude an open reading frame (ORF) for a protein operably linked on the5′ side or the 3′-side of the sequence encoding the nucleic acidmolecule; and/or an intron (intervening sequences).

Transcription of the nucleic acid molecule sequences can be driven froma promoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II(pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters are expressed at high levels in all cells; the levelsof a given pol II promoter in a given cell type depends on the nature ofthe gene regulatory sequences (enhancers, silencers, etc.) presentnearby. Prokaryotic RNA polymerase promoters are also used, providingthat the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells (Elroy et al., 1990, PNAS, 87:6743-47; Gao et al.,1993, Nucleic Acids Res 21:2867-72; Lieber et al., 1993, MethodsEnzymol., 217:47-66; Zhou et al., 1990, Mol. Cell. Biol. 10:4529-37).Several investigators have demonstrated that nucleic acid moleculesexpressed from such promoters can function in mammalian cells (e.g.Kashani-Sabet et al., 1992, Antisense Res. Dev., 2:3-15; Ojwang et al.,1992, PNAS 89:10802-06; Chen et al., 1992, Nucleic Acids Res.,20:4581-89; Yu et al., 1993, PNAS, 90:6340-44; L'Huillier et al., 1992,EMBO J., 11:4411-18; Lisziewicz et al., 1993, PNAS 90: 8000-04; Thompsonet al., 1995, Nucleic Acids Res., 23:2259; Sullenger et al., 1993,Science, 262:1566). More specifically, transcription units such as theones derived from genes encoding U6 small nuclear (snRNA), transfer RNA(tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res. 22:2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4:45; Beigelman et al., Int'lPCT Publication No. WO 96/18736. The above nucleic acid transcriptionunits can be incorporated into a variety of vectors for introductioninto mammalian cells, including but not restricted to, plasmid DNAvectors, viral DNA vectors (such as adenovirus or adeno-associated virusvectors), or viral RNA vectors (such as retroviral or alphavirusvectors) (see Couture and Stinchcomb, 1996 supra).

Nucleic acid molecule may be expressed within cells from eukaryoticpromoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarryand Lindquist, 1986, PNAS 83, 399; Scanlon et al., 1991, PNAS88:10591-95; Kashani-Sabet et al., 1992, Antisense Res. Dev., 2:3-15;Dropulic et al., 1992, J. Virol., 66:1432-41; Weerasinghe et al., 1991,J. Virol., 65:5531-34; Ojwang et al., 1992, PNAS, 89:10802-06; Chen etal., 1992, Nucleic Acids Res., 20:4581-89; Sarver et al., 1990 Science,247:1222-25; Thompson et al., 1995, Nucleic Acids Res., 23:2259; Good etal., 1997, Gene Therapy, 4:45. Those skilled in the art realize that anynucleic acid can be expressed in eukaryotic cells from the appropriateDNA/RNA vector. The activity of such nucleic acids can be augmented bytheir release from the primary transcript by a enzymatic nucleic acid(Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595;Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27:15-6; Taira et al.,1991, Nucleic Acids Res., 19:5125-30; Ventura et al., 1993, NucleicAcids Res., 21:3249-55; Chowrira et al., 1994, J. Biol. Chem.,269:25856.

A viral construct packaged into a viral particle would accomplish bothefficient introduction of an expression construct into the cell andtranscription of dsRNA construct encoded by the expression construct.

Methods for oral introduction include direct mixing of RNA with food ofthe organism, as well as engineered approaches in which a species thatis used as food is engineered to express an RNA, then fed to theorganism to be affected. Physical methods may be employed to introduce anucleic acid molecule solution into the cell. Physical methods ofintroducing nucleic acids include injection of a solution containing thenucleic acid molecule, bombardment by particles covered by the nucleicacid molecule, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the nucleic acidmolecule.

Other methods known in the art for introducing nucleic acids to cellsmay be used, such as lipid-mediated carrier transport, chemical mediatedtransport, such as calcium phosphate, and the like. Thus the nucleicacid molecules may be introduced along with components that perform oneor more of the following activities: enhance RNA uptake by the cell,promote annealing of the duplex strands, stabilize the annealed strands,or other-wise increase inhibition of the target gene.

Dosages

The useful dosage to be administered and the particular mode ofadministration will vary depending upon such factors as the cell type,or for in vivo use, the age, weight and the particular animal and regionthereof to be treated, the particular nucleic acid and delivery methodused, the therapeutic or diagnostic use contemplated, and the form ofthe formulation, for example, suspension, emulsion, micelle or liposome,as will be readily apparent to those skilled in the art. Typically,dosage is administered at lower levels and increased until the desiredeffect is achieved.

When lipids are used to deliver the nucleic acid, the amount of lipidcompound that is administered can vary and generally depends upon theamount of nucleic acid being administered. For example, the weight ratioof lipid compound to nucleic acid is preferably from about 1:1 to about30:1, with a weight ratio of about 5:1 to about 15:1 being morepreferred.

A suitable dosage unit of nucleic acid molecules may be in the range of0.001 to 0.25 milligrams per kilogram body weight of the recipient perday, or in the range of 0.01 to 20 micrograms per kilogram body weightper day, or in the range of 0.01 to 10 micrograms per kilogram bodyweight per day, or in the range of 0.10 to 5 micrograms per kilogrambody weight per day, or in the range of 0.1 to 2.5 micrograms perkilogram body weight per day.

Suitable amounts of nucleic acid molecules may be introduced and theseamounts can be empirically determined using standard methods. Effectiveconcentrations of individual nucleic acid molecule species in theenvironment of a cell may be about 1 femtomolar, about 50 femtomolar,100 femtomolar, 1 picomolar, 1.5 picomolar, 2.5 picomolar, 5 picomolar,10 picomolar, 25 picomolar, 50 picomolar, 100 picomolar, 500 picomolar,1 nanomolar, 2.5 nanomolar, 5 nanomolar, 10 nanomolar, 25 nanomolar, 50nanomolar, 100 nanomolar, 500 nanomolar, 1 micromolar, 2.5 micromolar, 5micromolar, 10 micromolar, 100 micromolar or more.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

Pharmaceutical compositions that include the nucleic acid moleculedisclosed herein may be administered once daily, qid, tid, bid, QD, orat any interval and for any duration that is medically appropriate.However, the therapeutic agent may also be dosed in dosage unitscontaining two, three, four, five, six or more sub-doses administered atappropriate intervals throughout the day. In that case, the nucleic acidmolecules contained in each sub-dose may be correspondingly smaller inorder to achieve the total daily dosage unit. The dosage unit can alsobe compounded for a single dose over several days, e.g., using aconventional sustained release formulation which provides sustained andconsistent release of the dsRNA over a several day period. Sustainedrelease formulations are well known in the art. The dosage unit maycontain a corresponding multiple of the daily dose. The composition canbe compounded in such a way that the sum of the multiple units of anucleic acid together contain a sufficient dose.

Pharmaceutical Compositions, Kits, and Containers

Also provided are compositions, kits, containers and formulations thatinclude a nucleic acid molecule (e.g., an siNA molecule) as providedherein for reducing expression of hsp47 for administering ordistributing the nucleic acid molecule to a patient. A kit may includeat least one container and at least one label. Suitable containersinclude, for example, bottles, vials, syringes, and test tubes. Thecontainers can be formed from a variety of materials such as glass,metal or plastic. The container can hold amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), cell population(s) and/orantibody(s). In one embodiment, the container holds a polynucleotide foruse in examining the mRNA expression profile of a cell, together withreagents used for this purpose. In another embodiment a containerincludes an antibody, binding fragment thereof or specific bindingprotein for use in evaluating hsp47 protein expression cells andtissues, or for relevant laboratory, prognostic, diagnostic,prophylactic and therapeutic purposes; indications and/or directions forsuch uses can be included on or with such container, as can reagents andother compositions or tools used for these purposes. Kits may furtherinclude associated indications and/or directions; reagents and othercompositions or tools used for such purpose can also be included.

The container can alternatively hold a composition that is effective fortreating, diagnosis, prognosing or prophylaxing a condition and can havea sterile access port (for example the container can be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The active agents in the composition can be a nucleicacid molecule capable of specifically binding hsp47 and/or modulatingthe function of hsp47.

A kit may further include a second container that includes apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution and/or dextrose solution. It can further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, stirrers, needles, syringes, and/orpackage inserts with indications and/or instructions for use.

The units dosage ampoules or multidose containers, in which the nucleicacid molecules are packaged prior to use, may include an hermeticallysealed container enclosing an amount of polynucleotide or solutioncontaining a polynucleotide suitable for a pharmaceutically effectivedose thereof, or multiples of an effective dose. The polynucleotide ispackaged as a sterile formulation, and the hermetically sealed containeris designed to preserve sterility of the formulation until use.

The container in which the polynucleotide including a sequence encodinga cellular immune response element or fragment thereof may include apackage that is labeled, and the label may bear a notice in the formprescribed by a governmental agency, for example the Food and DrugAdministration, which notice is reflective of approval by the agencyunder Federal law, of the manufacture, use, or sale of thepolynucleotide material therein for human administration.

Federal law requires that the use of pharmaceutical compositions in thetherapy of humans be approved by an agency of the Federal government. Inthe United States, enforcement is the responsibility of the Food andDrug Administration, which issues appropriate regulations for securingsuch approval, detailed in 21 U.S.C. §301-392. Regulation for biologicmaterial, including products made from the tissues of animals isprovided under 42 U.S.C. §262. Similar approval is required by mostforeign countries. Regulations vary from country to country, butindividual procedures are well known to those in the art and thecompositions and methods provided herein preferably comply accordingly.

The dosage to be administered depends to a large extent on the conditionand size of the subject being treated as well as the frequency oftreatment and the route of administration. Regimens for continuingtherapy, including dose and frequency may be guided by the initialresponse and clinical judgment. The parenteral route of injection intothe interstitial space of tissues is preferred, although otherparenteral routes, such as inhalation of an aerosol formulation, may berequired in specific administration, as for example to the mucousmembranes of the nose, throat, bronchial tissues or lungs.

As such, provided herein is a pharmaceutical product which may include apolynucleotide including a sequence encoding a cellular immune responseelement or fragment thereof in solution in a pharmaceutically acceptableinjectable carrier and suitable for introduction interstitially into atissue to cause cells of the tissue to express a cellular immuneresponse element or fragment thereof, a container enclosing thesolution, and a notice associated with the container in form prescribedby a governmental agency regulating the manufacture, use, or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofmanufacture, use, or sale of the solution of polynucleotide for humanadministration.

Indications

The nucleic acid molecules disclosed herein can be used to treatdiseases, conditions or disorders associated with hsp47, such as liverfibrosis, cirrhosis, pulmonary fibrosis, kidney fibrosis, peritonealfibrosis, chronic hepatic damage, and fibrillogenesis and any otherdisease or conditions that are related to or will respond to the levelsof hsp47 in a cell or tissue, alone or in combination with othertherapies. As such, compositions, kits and methods disclosed herein mayinclude packaging a nucleic acid molecule disclosed herein that includesa label or package insert. The label may include indications for use ofthe nucleic acid molecules such as use for treatment or prevention ofliver fibrosis, peritoneal fibrosis, kidney fibrosis and pulmonaryfibrosis, and any other disease or conditions that are related to orwill respond to the levels of hsp47 in a cell or tissue, alone or incombination with other therapies. A label may include an indication foruse in reducing expression of hsp47. A “package insert” is used to referto instructions customarily included in commercial packages oftherapeutic products, that contain information about the indications,usage, dosage, administration, contraindications, other therapeuticproducts to be combined with the packaged product, and/or warningsconcerning the use of such therapeutic products, etc.

Those skilled in the art will recognize that other anti-fibrosistreatments, drugs and therapies known in the art can be readily combinedwith the nucleic acid molecules herein (e.g. siNA molecules) and arehence contemplated herein.

The methods and compositions provided herein will now be described ingreater detail by reference to the following non-limiting examples.

Example 1 Preparation of siRNA for Gp46

Among optimal sequences for siRNA recognition in targeting a basesequence of HSP47, which is a common molecular chaperone for collagens(types I to IV), Sequences A and B were prepared in accordance with ansiRNA oligo design program by iGENE Therapeutics, Inc. Sequence C wasprepared by searching on the Internet using the siRNA Target Finder fromAmbion, Inc. and selecting 19 base sequences that would become a targetfor rat gp46 (human HSP47 homologue, GenBank Accession No. M69246). Whencarrying out the design, care was taken in starting at 75 to 100 basesdownstream from the initiation codon, positioning the first AA dimer,and making sure that the GC content was 30% to 70%. In this example,siRNAs having the sequences below were prepared.

A: GUUCCACCAUAAGAUGGUAGACAAC B: CCACAAGUUUUAUAUCCAAUCUAGCC: GAAACCUGUAGAGGCCGCA

Example 2 Inhibition of Gp46 Expression by Prepared siRNA

Normal rat kidney cells (NRK cells), which had rat gp46 and werefibroblasts producing collagen, were transfected with 0.1 nM to 50 nMsiRNA and cultured for 12 to 48 hours (FIG. 1). The amount of expressionof gp46 was checked by the western blot method (FIGS. 2 to 4, upper bandcorresponding to gp46, lower band corresponding to actin control). Allof the siRNAs inhibited the expression of gp46 protein remarkablycompared with a vehicle (FIG. 2). In the experiment below, siRNASequence A, which showed the strongest effect, was used. Inhibition bysiRNA was concentration dependent (FIG. 3); protein expression by gp46was about 90% inhibited by 50 nM siRNA at 48 hours (FIG. 4).

Example 3 Inhibition of Collagen Synthesis by Prepared siRNA

In order to examine the amount of collagen synthesized, ³H-proline wasadded to the culture supernatant of rat fibroblasts (NRK cells) underthe above-mentioned conditions (siRNA concentration 50 nM, time 48hours), and after transfection the amount of ³H in secreted protein wasexamined (FIG. 5). The amount of collagen synthesized was calculatedfrom the ratio of protein secreted in the supernatant to proteindegraded by collagenase when culturing gp46siRNA-transfected fibroblastsin the presence of ³H-proline in accordance with Peterkofsky et al.,1971 Biochemistry 10:988-94.

Collagen synthesis ratio=collagenase−sensitivefraction×100(5.4×collagenase−insensitive fraction+collagenase−sensitivefraction)

The collagen synthesis ratio in rat fibroblasts decreased by about 40%compared with a control group (FIG. 6).

Example 4 Specific Transfection of Nucleic Acid into HSC

An emulsion (VA-Lip-GFP) was prepared by mixing GFP expression plasmidand liposome-encapsulated VA formed by mixing 10% Vitamin A (VA) andliposome. Cationic liposomes containingO,O′-ditetradecanolyl-N-(α-trimethylammonioacetyl) diethanolaminechloride (DC-6-14) as a cationic lipid, cholesterol, anddioleoylphosphatidylethanolamine at a molar ratio of 4:3:3 weredissolved in a chloroform-methanol mixture (4:1, v:v), and the solventwas removed by evaporation under vacuum. The lipids were mixed with anaqueous 9% sucrose solution and hydrated at 60° C. and homogenized touniformity. The dispersion was extruded twice through apolyvinylidenedifluroide membrane filter with a 0.22 μm pore size. Thedispersion was aliquotted into glass vials and frozen and thenlyophilized. The dried liposomes were reconsitituted with distilledwater at a concentration of 1 mM DC-6-14 under vortexing before use.Specifically, 25 mg of Vitamin A was first dissolved in 87 μL of DMSOthus to give a 100 mM stock solution. To prepare VA-coupled liposomes,200 nmol of VA dissolved in DMSO was mixed with 100 nmole of DC-6-14 byvortexing at room temperature. The VA-siRNA-liposomes were intraportallyadministered to a rat, hepatic tissue was collected and fixed. Theemulsion was prepared by supposing that the amount of plasma for a 200 grat was about 10 mL, and setting the concentrations of VA and GFP inportal blood at 10 μM. 1 μL of this VA stock solution was mixed with 10μL of the VA-liposomes and 179 μL of PBS, 10 μg of GFP expressionplasmid was further added thereto to give a total of 200 μL, and themixture was vortexed for three minutes to give VA-Lip-GFP. The abdomenof an SD rat was opened, and the VA-Lip-GFP was slowly injected into aperipheral portal vein. Forty-eight hours after the injection, hepatictissue was harvested. Since compared with other hepatic cellsintermediate filament desmin is specifically expressed in HSC, whenfixed hepatic tissue was stained with Alexa Fluor 568-labeledanti-desmin antibody, and a fluorescence double image with GFP wasexamined, it was confirmed that GFP was expressed within the HSC (FIG.7). For untreated controls and a group to which the GFP expressionplasmid vector alone was administered, expression in rat HSC was notobserved, but in a group to which VA-Lip-GFP was administered,expression of GFP was observed specifically in stellate cells.

Example 5 Quantitative Analysis of Nucleic Acid Transfection Rate

In the same manner as in Example 4, except that FITC-labeled gp46siRNAwas used instead of the GFP expression plasmid, an emulsion(VA-Lip-gp46siRNA (FITC)) containing VA-encapsulated liposome andFITC-labeled gp46siRNA was prepared. A solution of siRNAgp46 (580pmole/μl in distilled water) was added to the VA-coupled liposomesolution of Example 4 with stirring at room temperature. The ratio ofsiRNA to DC-6-14 was 1:11.5 (mol/mol) and the siRNA to liposome ratio(wt/wt) was 1:1. Free VA or siRNA was removed by micropartition usingVIVASPIN concentrator, 30K MWCO, by three passes. Material trapped onthe membrane was reconstituted with PBS and intraportally administeredto an SD rat (10 μg as the amount of siRNA/200 μL). Forty-eight hoursafter administration hepatic tissue was harvested, aSMA (smooth muscleactin), which compared with other hepatic cells is expressedspecifically in HSC, was stained with Alexa Fluor 568-labeled anti-aSMAantibody, cell nuclei were stained with DAPI, and a fluorescence imagewas examined by a confocal laser scanning microscope (LSM). As shown onthe left-hand side of FIG. 8, in a group to which VA-Lip-gp46siRNA(FITC) was administered, a large number of cells emitting both greenfluorescence due to FITC and red fluorescence due to Alexa Fluor 568were observed, and when a quantitative analysis was carried out by NIHImage (the number of cells was counted by selecting any 10 fields from a×1000 fluorescence microscope photograph), the transfection efficiencywas 77.6% (average of 10 fields). On the other hand, in a group to whichLip-gp46siRNA (FITC) containing no VA was administered, the transfectionefficiency was a low value of 14.0% and, moreover, transfection intocells other than stellate cells was observed at 3.0% (right-hand side ofFIG. 8). It has been found from the results above that the transfectionefficiency into stellate cells is increased remarkably by including VA.

Example 6 Inhibition of Expression of Gp46 by VA-Lip-gp46siRNA

With regard to another section of the tissue harvested in Example 5,gp46 was stained with Alexa Fluor 568-labeled anti-HSP47 antibody andcell nuclei were stained with DAPI, and a fluorescence image wasexamined by a confocal laser scanning microscope. As shown in FIG. 9, itwas observed that in a group to which VA-Lip-gp46siRNA was administered,expression of gp46, which can be observed as a red fluorescence(right-hand side in the figure), was markedly reduced compared with acontrol group to which was administered VA-Lip-random siRNA containingrandom siRNA, which was not specific to gp46 (left-hand side in thefigure). The expression inhibition rate relative to an average of sixfields of the control group was 75%, which was extremely high, when thenumber of gp46-negative cells was examined by selecting any ten fieldsfrom a ×1000 fluorescence microscope photograph using NIH Image in thesame manner as in Example 7.

Example 7 Treatment of LC Rat (Intraportal Administration 1)

In accordance with a report by Jezequel et al. (Jezequel et al., 1987, JHepatol. 5:174-81), an LC model rat was prepared usingdimethylnitrosamine (DMN) (FIG. 10). Specifically, a 1 mL/kg dose of 1%DMN (intraperitoneal administration) was administered to a five week-oldSD rat (male) three straight days per week. As already reported, anincrease in fiber was observed from the 2nd week, and in the 4th weekthis was accompanied by the findings of marked fibrosis, destruction ofhepatic lobule structure, and formation of regenerative nodules beingobserved (FIG. 11). Then, by the same method as in Examples 4 and 5, anemulsion (VA-Lip-gp46siRNA) was prepared by formulating gp46siRNA as aliposome and mixing with 10% VA, and was administered. Administration ofVA-Lip-gp46siRNA was started in the 3rd week, by which time sufficientfibrosis was observed, and evaluation was carried out in the 4th and 5thweeks. Since it was confirmed by Example 2 that the effects wereobserved for up to 48 hours in vitro, administration was carried outtwice a week (FIG. 11). The amount administered was determined inaccordance with a report in which siRNA was directly injected (McCafferyet al., 2002, Nature 418: 38-39), and was 40 μg as the total amount ofsiRNA. From azan staining of the liver after administration of siRNA, inthe 4th week there was no apparent difference between a group to whichsaline had been administered, a group to which siRNA (random) had beenadministered, and a group to which siRNA (gp46) had been administered,but in the 5th week a decrease in the amount of fiber was observed forthe group to which gp46siRNA had been administered (FIG. 12). In orderto quantitatively analyze the amount of fiber, an unstained portion wasextracted using NIH Image, its area was measured (FIG. 13), and asignificant decrease in the area of collagen was observed for the groupto which gp46siRNA had been administered (FIG. 14). Furthermore, inorder to evaluate the degree of fibrosis using another measure, theamount of hydroxyproline, which is an indicator for fibrosis, wasquantitatively measured by a standard method. Specifically, after 20 mgof freeze-dried hepatic tissue was hydrolyzed with HCl for 24 hours, thereaction liquid was centrifuged, and the supernatant was treated with areagent such as Ehrlich's solution and centrifuged. The supernatant wasrecovered, and the amount of hydroxyproline in the hepatic tissue wasmeasured by measuring the absorbance at 560 nm (Hepatology 1998,28:1247-52). As shown in FIG. 15, in the group to which gp46siRNA hadbeen administered, the amount of hydroxyproline became very small.

Example 8 Treatment of LC Rat (Intraportal Administration 2)

Furthermore, in order to examine a change in the survival rate byadministration of the medicine of the present description, in accordancewith a method by Qi Z et al. (PNAS 1999; 96:2345-49), an LC model ratwas prepared using DMN in an amount that was increased by 20% over thenormal amount. In this model, a total of four intraportaladministrations were carried out in the first and second weeks.Administration details were: PBS, Lip-gp46siRNA, VA-Lip-random siRNA,and VA-Lip-gp46siRNA (n=7 for each group). After the third week, all ofthe controls (the group to which PBS had been administered, the group towhich VA-Lip-random siRNA had been administered, and the group to whichLip-gp46siRNA had been administered) were dead, but 6 out of 7 survivedfor the group to which VA-Lip-gp46siRNA had been administered (FIG. 16).Furthermore, in azan staining of the liver on the 21st day, an apparentdecrease in the amount of fiber was observed for the group to whichgp46siRNA had been administered (FIG. 17).

Example 9 Treatment of LC Rat (Intraportal Administration 3)

In another experiment, intraportal administration was carried out fromthe 3rd week for LC model rats (1% DMN 1 mg/kg intraperitoneallyadministered 3 times a week) prepared in accordance with the method byQi et al. and a method by Ueki et al., 1999, Nat Med. 5:226-30, as shownin Table 2 below (n=6 for each group). PBS was added to each substanceto be administered so as to make a total volume of 200 μL, and thefrequency of administration was once a week.

TABLE 2 Treatment Content of Frequency of group Administration DosageAdministration 10-1 VA VA 200 nmol Twice a week 10-2 10-2 Lip-gp46siRNAliposome 100 nmol; gp46siRNA 100 μg 10-3 VA-Lip-random VA 200 nmol;siRNA siRNA liposome 100 nmol; random- 100 μg 10-4 VA-Lip-gp46siRNA VA200 nmol; liposome 100 nmol; gp46siRNA 100 μg 10-5 PBS 200 μL Threetimes 10-6 VA VA 200 nmol a week 10-7 VA-Lip VA 200 nmol; liposome 100nmol 10-8 Lip-gp46siRNA liposome 100 nmol; gp46siRNA 150 μg 10-9VA-Lip-random VA 200 nmol; siRNA liposome 100 nmol; random-siRNA 150 μg10-10 VA-Lip-gp46siRNA VA 200 nmol; liposome 100 nmol; gp46siRNA 150 μg

From the results, in the groups other than the group to which themedicine of the present description had been administered (treatmentgroup 9-4), all 6 rats were dead by the 45th day after startingadministration of DMN, but in the group to which the medicine of thepresent description had been administered, all of the individuals apartfrom one case, which was dead on the 36th day, survived for more than 70days after starting administration of DMN (FIG. 18). For the deadindividuals, the amount of hepatic fiber was quantitatively analyzedbased on the area of collagen in the same manner as in Example 7, andthe increase in the amount of hepatic fiber was remarkably inhibited byadministration of VA-Lip-gp46siRNA (FIG. 19).

Example 10 Treatment of LC Rat (Intravenous Administration)

Intravenous administration was carried out from the 3rd week for LCmodel rats (1% DMN 1 μg/BW (g) intraperitoneally administered 3 times aweek) prepared in the same manner as in Example 9, as shown in the tablebelow (n=6 for each group). PBS was added to each substance to beadministered so as to make a total volume of 200 pt. The administrationperiod was up to death except that it was up to the 7th week for Group10-4 and the 6th week for Group 10-10.

From the results, in the groups other than the groups to which themedicine of the present description had been administered (treatmentgroups 10-4 and 10-10), all 6 rats were dead by the 45th day afterstarting administration of DMN, but in the groups to which the medicineof the present description had been administered, all of theindividuals, apart from a case in which two rats were dead on the 45thday in treatment group 10-4, survived for more than 70 days afterstarting administration of DMN (FIGS. 20 and 21). For the deadindividuals, the amount of hepatic fiber was quantitatively analyzed inthe same manner as in Example 7, and the increase in the amount ofhepatic fiber was remarkably inhibited by administration ofVA-Lip-gp46siRNA (FIG. 22).

The above-mentioned results show that the medicine of the presentdescription is extremely effective for the prevention and treatment offibrosis, in which stellate cells are involved.

Example 11 Improvement of Results by RBP (Retinol-Binding Protein)

The influence of RBP on VA-Lip-gp46siRNA transfection efficiency wasexamined using LI90, a cell line derived from human HSC. 100 nM ofVA-Lip-gp46siRNA (FITC) prepared in Example 5, together with variousconcentrations (i.e. 0, 0.1, 0.5, 1, 2, 4, or 10%) of FBS (fetal bovineserum), were added to LI90 during culturing and incubated for 48 hours,a fluorescence image was observed by LSM, and the amount of siRNAincorporated into individual cells was quantitatively analyzed by FACS.FBS contained about 0.7 mg/dL of RBP. As shown in FIG. 23, FBS (RBP)gave a concentration-dependent increase in the amount of siRNAtransfection. Subsequently, 100 nM of VA-Lip-gp46siRNA (FITC) and 4%FBS, together with 10 μg (21.476 nmol) of anti-RBP antibody, were addedto LI90 during culturing, and the siRNA transfection efficiency wasevaluated in the same manner. As shown in FIG. 24, the increase in theamount of transfection by RBP was markedly decreased by the addition ofanti-RBP antibody. The above-mentioned results show that RBP iseffective in further enhancing transfection of the medicine of thepresent description.

Example 12 Selecting Hsp47 Nucleic Acid Molecule Sequences

Nucleic acid molecules (e.g., siNA ≦25 nucleotides) against Hsp47 weredesigned using several computer programs including siRNA at WhiteheadInstitute for Biomedical Research, siRNA Design (Integrated DNATechnologies), BLOCK-iT RNAi Designer (Invitrogen), siDESIGN Center(Dharmacon), and BIOPREDsi (Friedrich Miescher Institute for BiomedicalResearch). The sequences of top scored siRNAs from these programs werecompared and selected (see Table 3) based on the algorithms as well asthe sequence homology between human and rat. Candidate sequences werevalidated by in vitro knocking down assays.

Several parameters were considered for selecting a nucleic acid molecule(e.g., a 21-mer siRNA) sequence. Exemplary parameters include:

-   -   thermodynamic stability (RISC favors the strand with less stable        5′-end)    -   30-52% GC content    -   positional nucleotide preference:

(C/G)₁NNNNNNNN(A/U)₁₀NNNNNNNN(A/U)₁₉

-   -   -   where N is any nucleotide

    -   devoid of putative immunostimulatory motifs

    -   2-nucleotide 3′ overhang

    -   position of siRNA within the transcript (preferably within cDNA        region)

    -   sequence specificity (checked by using BLAST)

    -   variations in single nucleotide by checking SNP database

iRNA sequences having ≦25 nucleotides were designed based on theforegoing methods. Corresponding Dicer substrate siRNA (e.g., ≧26nucleotides) were designed based on the smaller sequences and extend thetarget site of the siNA ≦25 nucleotide by adding four bases to the3′-end of the sense strand and 6 bases to the 5′-end of the antisensestrand. The Dicer substrates that were made generally have a 25 basesense strand a 27 base antisense strand with an asymmetric blunt endedand 3′-overhang molecule. The sequences of the sense and the anti-sensestrand without base modification (base sequence) and with modifications(experimental sequence) are provided in Table 3.

TABLE 3 Base sequence Experimental sequence Target (corresponding(corresponding siRNA region nucleotides of SEQ ID NO: 1)nucleotides of SEQ ID NO: 1) siHSP47-C human/rat sense 5′GGACAGGCCUCUACAACUAUU (SEQ ID 5′ GGACAGGCCUCUACAACUAdTdT (SEQ hsp47NO: 3) ID NO: 5) [945-963] [945-963] anti- 5′UAGUUGUAGAGGCCUGUCCUU (SEQ ID 5′ UAGUUGUAGAGGCCUGUCCdTdT (SEQ senseNO: 4) ID NO: 6) [945-963] [945-963] siHSP47- human/rat sense 5′GGACAGGCCUCUACAACUACUACGA 5′ GGACAGGCCUCUACAACUACUACdGdA Cd hsp47(SEQ ID NO: 7) (SEQ ID NO: 9) [945-969] [945-969] anti-5′UCGUAGUAGUUGUAGAGGCCUGUCCUU 5′UCGUAGUAGUUGUAGAGGCCUGUCCUU sense(SEQ ID NO: 8) (SEQ ID NO: 10) [945-969] [945-969] siHSP47-1 human/ratsense 5′ CAGGCCUCUACAACUACUAUU (SEQ ID 5′ CAGGCCUCUACAACUACUAdTdT (SEQhsp47 NO: 11) ID NO: 13) [948-966] [948-966] anti- 5′UAGUAGUUGUAGAGGCCUGUU (SEQ ID 5′ UAGUAGUUGUAGAGGCCUGdTdT (SEQ senseNO: 12) ID NO: 14) [948-966] [948-966] siHSP47- human sense 5′CAGGCCUCUACAACUACUACGACGA 5′ CAGGCCUCUACAACUACUACGACdGdA 1d hsp47(SEQ ID NO: 15) (SEQ ID NO: 17) [948-972] [948-972] anti- 5′CGUCGUAGUAGUUGUAGAGGCCUGUU 5′ sense (SEQ ID NO: 16)CAGCUUUUCCUUCUCGUCGUCGUAGUU [948-972] (SEQ ID NO: 2724) [948-972]siHsp47-2 human sense 5′ GAGCACUCCAAGAUCAACUUU (SEQ ID 5′GAGCACUCCAAGAUCAACUdTdT (SEQ hsp47 NO: 19) ID NO: 21) [698-717][698-717] anti- 5′ AGUUGAUCUUGGAGUGCUCUU (SEQ ID 5′AGUUGAUCUUGGAGUGCUCdTdT (SEQ sense NO: 20) ID NO: 22) [698-716][698-716] siHsp47- human sense 5′ GAGCACUCCAAGAUCAACUUCCGCG 5′GAGCACUCCAAGAUCAACUUCCGdCdG 2d hsp47 (SEQ ID NO: 23) (SEQ ID NO: 25)[698-722] [698-722] anti- 5′CGCGGAAGUUGAUCUUGGAGUGCUCUU 5′GCGGAAGUUGAUCUUGGAGUGCUCUU sense (SEQ ID NO: 24) (SEQ ID NO: 26)[698-722] [698-722] siHsp47- rat Gp46 sense 5′ GAACACUCCAAGAUCAACUUCCGAG5′ GAACACUCCAAGAUCAACUUCCGdAdG 2d rat (SEQ ID NO: 27) (SEQ ID NO: 29)[587-611] [587-611] anti- 5′CUCGGAAGUUGAUCUUGGAGUGUUCUU 5′UCGGAAGUUGAUCUUGGAGUGUUCUU sense (SEQ ID NO: 28) (SEQ ID NO: 30)[587-611] [587-611] siHsp47-3 human sense 5′CUGAGGCCAUUGACAAGAAUU (SEQ ID 5′ CUGAGGCCAUUGACAAGAAdTdT (SEQ hsp47NO: 31) ID NO: 33) [1209-1227] [1209-1227] anti- 5′UUCUUGUCAAUGGCCUCAGUU (SEQ ID 5′ UUCUUGUCAAUGGCCUCAGdTdT (SEQ senseNO: 32) ID NO: 34) [1209-1227] [1209-1227] siHsp47- human sense 5′CUGAGGCCAUUGACAAGAACAAGGC 5′ CUGAGGCCAUUGACAAGAACAAGdGdC 3d hsp47(SEQ ID NO: 35) (SEQ ID NO: 37) [1209-1233] [1209-1233] anti- 5′CCUUGUUCUUGUCAAUGGCCUCAGUU 5′GCCUUGUUCUUGUCAAUGGCCUCAGUU sense(SEQ ID NO: 36) (SEQ ID NO: 38) [1209-1233] [1209-1233] siHsp47-4 humansense 5′ CUACGACGACGAGAAGGAAUU (SEQ ID 5′ CUACGACGACGAGAAGGAAdTdT (SEQhsp47 NO: 39) ID NO: 41) [964-982] [964-982] anti- 5′UUCCUUCUCGUCGUCGUAGUU (SEQ ID 5′ UUCCUUCUCGUCGUCGUAGdTdT (SEQ senseNO: 40) ID NO: 42) [964-982] [964-982] siHsp47- human sense 5′CUACGACGACGAGAAGGAAAAGCUG 5′ CUACGACGACGAGAAGGAAAAGCdTdG 4d hsp47(SEQ ID NO: 43) (SEQ ID NO: 45) [964-988] [964-988] anti- 5′AGCUUUUCCUUCUCGUCGUCGUAGUU 5′ sense (SEQ ID NO: 44)CAGCUUUUCCUUCUCGUCGUCGUAGUU [964-988] (SEQ ID NO: 2727) [964-988]siHsp47-5 human sense 5′ GCCACACUGGGAUGAGAAAUU (SEQ ID 5′GCCACACUGGGAUGAGAAAdTdT (SEQ hsp47 NO: 47) ID NO: 49) [850-870][850-870] anti- 5′ UUUCUCAUCCCAGUGUGGCUU (SEQ ID 5′UUUCUCAUCCCAGUGUGGCdTdT (SEQ sense NO: 48) ID NO: 50) [850-868][850-868] siHsp47-6 human sense 5′ GCAGCAAGCAGCACUACAAUU (SEQ ID 5′GCAGCAAGCAGCACUACAAdTdT (SEQ hsp47 NO: 51) ID NO: 53) [675-693][675-693] anti- 5′ UUGUAGUGCUGCUUGCUGCUU (SEQ ID 5′UUGUAGUGCUGCUUGCUGCdTdT (SEQ sense NO: 52) ID NO: 54) [675-693][675-693] siHsp47-7 human sense 5′ CCGUGGGUGUCAUGAUGAUUU (SEQ ID 5′CCGUGGGUGUCAUGAUGAUdTdT (SEQ hsp47 NO: 55) ID NO: 57) [921-939][921-939] anti- 5′ AUCAUCAUGACACCCACGGUU (SEQ ID 5′AUCAUCAUGACACCCACGGdTdT (SEQ sense NO: 56) ID NO: 58) [921-939][921-939]

Example 13

In order to screen for the potency of various siNA molecules againstboth the human and rat hsp47 genes, various reporter cell lines wereestablished by lenti-viral induction of human HSP47 cDNA-greenfluorescent protein (GFP) or rat GP46 cDNA-GFP construct into 293,HT1080, human HSC line hTERT, or NRK cell lines. These cell lines werefurther evaluated by siRNA against GFP. The remaining fluorescencesignal was measured and normalized to scrambled siRNA (Ambion) andsubsequently normalized to cell viability. The results showed that siRNAagainst GFP knocks down the fluorescence to different extent indifferent cell lines (FIG. 25). 293_HSP47-GFP and 293_GP46-GFP celllines were selected for siHsp47 screening due to their ease oftransfection and sensitivity to fluorescence knockdown.

Cells were transfected with 1.5 pmol per well of siNA against GFP in96-well tissue culture plates using Lipofectamine RNAiMAX (Invitrogen)in a reverse transfection manner. Cells were seeded at 6,000 cells perwell and mixed with the siNA complexes. Fluorescence readings were takenafter 72 hours incubation on a Synergy 2 Multi-Mode Microplate Reader(BioTek).

Cells treated with or without siNA were measured for viability after 72hours incubation using CellTiter-Glo Luminescent Cell Viability AssayKit according to the manual (Promega). The readings were normalized tosamples treated with scrambled siNA molecules.

Example 14 Evaluation of Inhibitory Efficiency of siHsp47 on Hsp47Expression in Reporter Cell Lines

siNAs against hsp47 were evaluated for their inhibitory efficiency in293_HSP47-GFP and 293_GP46-GFP cell lines by evaluating the change influorescent signal from the reporter GFP. The experiments were carriedout as described in Example 2. The fluorescent signals were normalizedto fluorescent signals from cells treated with scrambled siRNA (Ambion)which served as a control. The results indicate that the tested hsp47siNA molecules were effective in inhibiting hsp47 mRNA in both celllines. However, siNA against GP46 mRNA (as published in the 2008 Sato etal paper) was effective only in the 293_GP46-GFP cell line. The resultsare shown in FIGS. 26A and 26B.

The 293_HSP47-GFP and 293_GP46-GFP cell lines treated with siRNA againsthsp47 and gp46 were evaluated for viability using the methods describedin Example 2. The cell viability was normalized to cells treated withscrambled siRNA (Ambion). The results indicate that the cell viabilitywas not affected significantly by the treatment with siNA molecules.However, the cell viability of 293_HSP47-GFP cell lines treated withdifferent hsp47 siNA molecules varied depending on the siNA moleculesused, while the viability of 293_GP46-GFP cell lines were similar.Viability for 293 HSP47-GFP cells was lower for siHsp47-6 and Hsp47-7treated cells than the rest. The results are shown in FIGS. 26C and 26D.

Example 15 Evaluation of siHsp47 Inhibitory Effect on Hsp47 mRNA byTAQMAN® qPCR

In Example 14, the knock down efficiency of siHsp47s in reporter celllines was evaluated by change in fluorescent signal. To validate theresults at the mRNA level, siRNAs targeting endogenous hsp47 weretransfected into cells of the human HSC cell line hTERT usingLipofectamine RNAiMAX (Invitrogen) in a reverse transfection manner asdescribed in Example 13.

The hsp47 mRNA level was evaluated for knock down efficiency of thevarious tested siHsp47 siNA molecules. Briefly, mRNA were isolated fromhTERT cells 72 hours after transfection using an RNeasy mini kit(Qiagen). The level of hsp47 mRNA was determined by reversetranscription coupled with quantitative PCR using TAQMAN® probes.Briefly, cDNA synthesis was carried out using High-Capacity cDNA ReverseTranscription Kit (ABI) according to the manufacturer's instruction, andsubjected to TaqMan Gene Expression Assay (ABI, hsp47). The level ofhsp47 mRNA was normalized to the level of GAPDH mRNA according to themanufacturer's instruction (ABI). The results indicate that siHsp47-Cwas the most effective among all the hsp47 siRNAs, siHsp47-2 andsiHsp47-2d were the next most effective. The combinations of siHsp47-1with siHsp47-2 or siHsp47-1 with siHsp47-2d were more effective thansiHsp47-1 alone. The results are shown in FIG. 27.

Example 16 Validation of siHsp47 Knock Down Effect at the Protein Level

The inhibitory effect of different Hsp47 siNA molecules (siHsp47) onhsp47 mRNA expression were validated at the protein level by measuringthe HSP47 in hTERT cells transfected with different siHsp47.Transfection of hTERT cells with different siHsp47 were performed asdescribed in Example 13. Transfected hTERT cells were lysed and the celllysate were clarified by centrifugation. Proteins in the clarified celllysate were resolved by SDS polyacrylamide gel electrophoresis. Thelevel of HSP47 protein in the cell lysate were determined using ananti-HSP47 antibody (Assay Designs) as the primary antibody, Goatanti-mouse IgG conjugated with HRP (Millipore) as the secondaryantibody, and subsequently detected by Supersignal West PicoChemiluminescence kit (Pierce). Anti-actin antibody (Abcam) was used asa protein loading control. The result showed significant decrease in thelevel of Hsp47 protein in cells treated with siHsp47-C, siHsp47-2d,alone or combination of siHsp47-1 with siHsp47-2d.

Example 17 Downregulation of Collagen I Expression by Hsp47 siRNA

To determine the effect of siHsp47 on collagen I expression level,collagen I mRNA level in hTERT cells treated with different siRNAagainst hsp47 was measured. Briefly, hTERT cells were transfected withdifferent siHsp47 as described in Example 13. The cells were lysed after72 hours and mRNA were isolated using RNeasy mini kit according to themanual (Qiagen). The level of collagen I mRNA was determined by reversetranscription coupled with quantitative PCR using TAQMAN® probes.Briefly, cDNA synthesis was carried out using High-Capacity cDNA ReverseTranscription Kit (ABI) according to the manual, and subjected to TaqManGene Expression Assay (ABI, COL1A1 assay). The level of collagen I mRNAwas normalized to the level of GAPDH mRNA according to themanufacturer's instruction (ABI). The signals were normalized to thesignal obtained from cells transfected with scrambled siNA. The resultindicated that collagen I mRNA level is significantly reduced in thecells treated with some of the candidates siHsp47-2, siHsp47-2d, andtheir combination with siHsp47-1 and shown in FIG. 28.

Example 18 Immunofluorescence Staining of Hsp47 siRNA Treated hTERTCells

To visualize the expression of two fibrosis markers, collagen I andalpha-smooth muscle actin (SMA), in hTERT cells transfected with orwithout siHsp47, the cells were stained with rabbit anti-collagen Iantibody (Abcam) and mouse anti-alpha-SMA antibody (Sigma). Alexa Fluor594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG(Invitrogen (Molecular Probes)) were used as secondary antibodies tovisualize collagen I (green) and alpha-SMA (red). Hoescht was used tovisualize nuleus (blue). The results indicate correlation between siRNAknocking down of some of the target genes and collagen/SMA expression.

Example 19 In Vivo Testing of siHSP47 in Animal Models of Liver Fibrosis

The siRNA duplex sequence for HSP47 (siHSP47C) is as listed below.

Sense (5′->3′) (SEQ ID NO: 2731) ggacaggccucuacaacuaTTAntisense (5′->3′) (SEQ ID NO: 2732) uaguuguagaggccuguccTT

10 mg/ml siRNA stock solution was prepared by dissolving in nucleasefree water (Ambion). For treatment of cirrhotic rats, siRNA wasformulated with vitamin A-coupled liposome as described by Sato et al(Sato Y. et al. 2008 Nature Biotech. 26:431) in order to targetactivated HSC that produce collagen. The vitamin A (VA)-liposome-siRNAformulation consists of 0.33 μmol/ml of VA, 0.33 μmol/ml of liposome(Coatsome EL, NOF Corporation) and 0.5 μg/μl of siRNA in 5% glucosesolution.

Four week-old male SD rats were induced with liver cirrhosis with 0.5%dimethylnitrosoamine (DMN) (Wako Chemicals, Japan) in phosphate-bufferedsaline (PBS). A dose of 2 ml/kg per body weight was administeredintraperitoneally for 3 consecutive days per week on days 0, 2, 4, 7, 9,11, 14, 16, 18, 21, 23, 25, 28, 30, 32, 34, 36, 38 and 40.

siRNA treatment: siRNA treatment was carried out from day 32 and for 5intravenous injections. In detail, rats were treated with siRNA onday-32, 34, 36, 38 and 40. Then rats were sacrificed on day-42 or 43. 3different siRNA doses (1.5 mg siRNA per kg body weight, 2.25 mg siRNAper kg body weight, 3.0 mg siRNA per kg body weight) were tested.

Detail of tested groups and number of animals in each group are asfollows:

1) Cirrhosis was induced by DMN injection, then 5% glucose was injectedinstead of siRNA) (n=10)

2) VA-Lip-siHSP47C 1.5 mg/kg (n=10)

3) VA-Lip-siHSP47C 2.25 mg/kg (n=10)

4) VA-Lip-siHSP47C 3.0 mg/Kg (n=10)

5) Sham (PBS was injected instead of DMN; 5% Glucose was injectedinstead of siRNA) (n=6)

6) No treatment control (Intact) (n=6)

VA-Lip refers to vitamin A—liposome complex.

Evaluation of therapeutic efficacy: On day 43, 2 out of 10 animals inthe “diseased rat” group and 1 out of 10 animals in “VA-Lip-siHSP47CsiRNA 1.5 mg/kg” died due to development of liver cirrhosis. Theremainder of the animals survived. After rats were sacrificed, livertissues were fixed in 10% formalin. Then, the left lobule of each liverwas embedded in paraffin for histology. Tissue slides were stained withSirius red, and hematoxylin and eosin (HE). Sirius red staining wasemployed to visualize collagen-deposits and to determine the level ofcirrhosis. HE staining was for nuclei and cytoplasm as counter-staining.Each slide was observed under microscope (BZ-8000, Keyence Corp. Japan)and percentage of Sirius red-stained area per slide was determined byimage analysis software attached to the microscope. At least 4 slidesper each liver were prepared for image analysis, and whole area of eachslide (slice of liver) was captured by camera and analyzed. Statisticalanalysis was carried out by Student's t-test.

Results: FIG. 29 shows the fibrotic areas. The area of fibrosis in“diseased rats” was higher than in the “sham” or “no treatment control”groups. Therefore, DMN treatment induced collagen deposition in theliver, which was a typical observation of liver fibrosis. However, thearea of fibrosis was significantly reduced by the treatment of siRNAtargeting HSP47 gene, compared with “disease rat” group (FIG. 29). Thisresult indicates that siRNA as disclosed herein has a therapeuticefficacy in actual disease.

Additional siRNA compounds are tested in the liver fibrosis animalmodel, and were shown to reduce the fibrotic area in the liver.

Example 20 Generation of Sequences for Active Double-Stranded RNACompounds to HSP47/SERPINH1 and Production of the siRNAs Shown in Tables4, 5, B, C, D and E

Duplexes are generated by annealing complementary single-strandedoligonucleotides. In a laminar flow hood, a 500 μM stock solution ofsingle-stranded oligonucleotide is prepared by diluting in WFI (waterfor injection). Actual ssRNA concentrations are determined by dilutingeach 500 μM ssRNA 1:200 using WFI, and measuring the OD using Nano Drop.The procedure is repeated 3 times and the average concentration iscalculated. The stock solution was then diluted to a final concentrationof 250 μM. Complementary single-strands were annealed by heating to 85°C. and allowing to cool to room temperature over at least 45 minutes.Duplexes were tested for complete annealing by testing 5 μl on a 20%polyacrylamide gel and staining. Samples were stored at −80° C.

Tables 4, 5, B, C, D and E provide siRNAs for HSP47/SERPINH1. For eachgene there is a separate list of 19-mer siRNA sequences, which areprioritized based on their score in the proprietary algorithm as thebest sequences for targeting the human gene expression.

The following abbreviations are used in the Tables 4, 5, B, C, D and Eherein: “other spec or Sp.” refers to cross species identity with otheranimals: D-dog, Rt-rat, Rb-Rabbit, Rh-rhesus monkey, P—Pig, M—Mouse;ORF: open reading frame. 19-mers (Tables 5, B, C), and 18+1-(Tables 4,D, E) mers refer to oligomers of 19 and 18+1 (U in position 1 ofAntisense, A in position 19 of sense strand) ribonucleic acids inlength, respectively.

siRNA oligonucleotides useful in generating double-stranded RNAmolecules are disclosed in Tables 4, 5, B, C, D and E below.

TABLE 4 SEQ SEQ ID ID NO NO Cross Ident Human Name SEN Sense (5′ > 3′)AS Antisense (5′ > 3′) Species gi″32454740 SERPINH1_2 60GAGACACAUGGGUGCUAUA 127 UAUAGCACCCAUGUGUCUC H, Rt, Rh, [1533-1551] M, D(18/19) SERPINH1_3 61 GGGAAGAUGCAGAAGAAGA 128 UCUUCUUCUGCAUCUUCCCH, Rt, Rh, [1112-1130] Rb (18/19) SERPINH1_5 62 GAAGAAGGCUGUUGCCAUA 129UAUGGCAACAGCCUUCUUC H, Rt [1123-1141] (18/19) SERPINH1_6 63ACAAGAUGCGAGACGAGUA 130 UACUCGUCUCGCAUCUUGU H, Rt, Rh, [1464-1482](18/19) SERPINH1_7 64 GGACAACCGUGGCUUCAUA 131 UAUGAAGCCACGGUUGUCCH, Rh, M [886-904] (18/19) SERPINH1_8 65 UGCAGUCCAUCAACGAGUA 132UACUCGUUGAUGGACUGCA H, Rt, Rh, M [738-756] (18/19) SERPINH1_9 66GCCUCAUCAUCCUCAUGCA 133 UGCAUGAGGAUGAUGAGGC H, Rt, Rh, [1026-1044] M, D(18/19) SERPINH1_10 67 CGCGCUGCAGUCCAUCAAA 134 UUUGAUGGACUGCAGCGCGH, Rt, Rh [733-751] (18/19) SERPINH1_11 68 CGGACAGGCCUCUACAACA 135UGUUGUAGAGGCCUGUCCG H, Rt, Rh, P [944-962] (18/19) SERPINH1_13 69UGACAAGAUGCGAGACGAA 136 UUCGUCUCGCAUCUUGUCA H, Rh [1462-1480] (18/19)SERPINH1_14 70 CCAGCCUCAUCAUCCUCAA 137 UUGAGGAUGAUGAGGCUGG H, M, Rt,[1023-1041] Rh, D- (18/19) SERPINH1_15 71 GCUGCAGUCCAUCAACGAA 138UUCGUUGAUGGACUGCAGC H, Rt, Rh [736-754] (18/19) SERPINH1_16 72GCAGCGCGCUGCAGUCCAA 139 UUGGACUGCAGCGCGCUGC H, Rt, Rh [729-747] (18/19)SERPINH1_17 73 UGAGACACAUGGGUGCUAA 140 UUAGCACCCAUGUGUCUCA H, Rt, Rh[1532-1550] M, D (18/19) SERPINH1_19 74 GGUGGAGGUGACCCAUGAA 141UUCAUGGGUCACCUCCACC H, Rt, Rh, M [1159-1177] (18/19) SERPINH1_20 75CUUUGACCAGGACAUCUAA 142 UUAGAUGUCCUGGUCAAAG H, Rt, Rh [1324-1342](18/19) SERPINH1_21 76 GGAGGUGACCCAUGACCUA 143 UAGGUCAUGGGUCACCUCCH, Rt, Rh, [1162-1180] M, D (18/19) SERPINH1_22 77 CUCCUGAGACACAUGGGUA144 UACCCAUGUGUCUCAGGAG H, D [1528-1546] (18/19) SERPINH1_23 78AGAAGAAGGCUGUUGCCAA 145 UUGGCAACAGCCUUCUUCU H, Rt [1122-1140] (18/19)SERPINH1_24 79 AGCUCUCCAGCCUCAUCAA 146 UUGAUGAGGCUGGAGAGCU H, Rt, D,[1017-1035] M, P, Rh (18/19) SERPINH1_25 80 CUGCAGUCCAUCAACGAGA 147UCUCGUUGAUGGACUGCAG H, Rt, Rh M [737-755] (18/19) SERPINH1_26 81CCGGACAGGCCUCUACAAA 148 UUUGUAGAGGCCUGUCCGG H, Rt, Rh, [943-961] Rb, P(18/19) SERPINH1_27 82 GCACCGGACAGGCCUCUAA 149 UUAGAGGCCUGUCCGGUGCH, Rt, Rh, [940-958] Rb, P (18/19) SERPINH1_28 83 GCAGAAGAAGGCUGUUGCA150 UGCAACAGCCUUCUUCUGC H, Rt [1120-1138] (18/19) SERPINH1_31 84AGAAGGCUGUUGCCAUCUA 151 UAGAUGGCAACAGCCUUCU H, Rt [1125-1143] (18/19)SERPINH1_32 85 AGCGCAGCGCGCUGCAGUA 152 UACUGCAGCGCGCUGCGCU H, Rt, Rh,[726-744] (18/19) SERPINH1_33 86 GACACAUGGGUGCUAUUGA 153UCAAUAGCACCCAUGUGUC H, Rt, Rh, M [1535-1553]  (18/19)| SERPINH1_34 87GGGCCUGACUGAGGCCAUA 154 UAUGGCCUCAGUCAGGCCC H, Rt [1201-1219] (18/19)SERPINH1_35 88 AGACACAUGGGUGCUAUUA 155 UAAUAGCACCCAUGUGUCU H, Rt, Rh, M[1534-1552] (18/19) SERPINH1_36 89 CCAUGACCUGCAGAAACAA 156UUGUUUCUGCAGGUCAUGG H, Rt, Rh, M [1171-1189] (18/19) SERPINH1_37 90AGAUGCAGAAGAAGGCUGA 157 UCAGCCUUCUUCUGCAUCU H, Rt, Rh, M [1116-1134](18/19) SERPINH1_38 91 CAAGCUCUCCAGCCUCAUA 158 UAUGAGGCUGGAGAGCUUGH, Rt, Rh, [1015-1033] M, P, D (18/19) SERPINH1_39 92UGCAGAAGAAGGCUGUUGA 159 UCAACAGCCUUCUUCUGCA H, Rt [1119-1137] (18/19)SERPINH1_41 93 CAGCCUCAUCAUCCUCAUA 160 UAUGAGGAUGAUGAGGCUG H, Rt, Rh,[1024-1042] M, D (18/19) SERPINH1_42 94 GACAGGCCUCUACAACUAA 161UUAGUUGUAGAGGCCUGUC H, Rt, Rh, [946-964] Rb, P (18/19) SERPINH1_43 95GAUGCAGAAGAAGGCUGUA 162 UACAGCCUUCUUCUGCAUC H, Rt, Rh, M [1117-1135](18/19) SERPINH1_44 96 ACCCAUGACCUGCAGAAAA 163 UUUUCUGCAGGUCAUGGGUH, Rt, Rh, M [1169-1187] (18/19) SERPINH1_45 97 ACUCCAAGAUCAACUUCCA 164UGGAAGUUGAUCUUGGAGU H, Rt, Rh, [702-720] M, D (18/19) SERPINH1_45a 98ACUCCAAGAUCAACUUCCU 165 AGGAAGUUGAUCUUGGAGU H, Rt, Rh, [702-720] M, D(18/19) SERPINH1_48 99 AGGCCUCUACAACUACUAA 166 UUAGUAGUUGUAGAGGCCUH, Rt, Rh, [949-967] Rb, P, D (18/19) SERPINH1_49 100CACUCCAAGAUCAACUUCA 167 UGAAGUUGAUCUUGGAGUG H, Rt, Rh, [701-719] M, D(18/19) SERPINH1_51 101 UCCUGAGACACAUGGGUGA 168 UCACCCAUGUGUCUCAGGAH, Rt, D, M [1529-1547] (18/19) SERPINH1_52 102 GACAAGAUGCGAGACGAGA 169UCUCGUCUCGCAUCUUGUC H, Rt, Rh, [1463-1481] (18/19) SERPINH1_53 103GGUGACCCAUGACCUGCAA 170 UUGCAGGUCAUGGGUCACC H, Rt, Rh, M [1165-1183](18/19) SERPINH1_59 104 CCGAGGUGAAGAAACCUGA 171 UCAGGUUUCUUCACCUCGGH, Rt, Rh, [285-303] (18/19) SERPINH1_51a 105 UCCUGAGACACAUGGGUGU 172ACACCCAUGUGUCUCAGGA H, Rt, D, M [1529-1547] (18/19) SERPINH1_61 106GCACUCCAAGAUCAACUUA 173 UAAGUUGAUCUUGGAGUGC H, Rh, D [700-718] (18/19)SERPINH1_62 107 GUGGUGGAGGUGACCCAUA 174 UAUGGGUCACCUCCACCAC H, Rt, Rh,[1157-1175] M, Rb (18/19) SERPINH1_64 108 GCCGAGGUGAAGAAACCUA 175UAGGUUUCUUCACCUCGGC H, Rt, Rh, [284-302] (18/19) SERPINH1_65 109GCUCUCCAGCCUCAUCAUA 176 UAUGAUGAGGCUGGAGAGC H, Rt, D, [1018-1036]M, P, Rh (18/19) SERPINH1_66 110 GAUGCACCGGACAGGCCUA 177UAGGCCUGUCCGGUGCAUC H, Rt, Rh, [937-955] M, Rb, P (18/19) SERPINH1_68111 CUCUCCAGCCUCAUCAUCA 178 UGAUGAUGAGGCUGGAGAG H, Rt, D, [1019-1037]M, P, Rh (18/19) SERPINH1_69 112 GCAGACCACCGACGGCAAA 179UUUGCCGUCGGUGGUCUGC H, Rt, D [763-781] (18/19) SERPINH1_70 113AGUCCAUCAACGAGUGGGA 180 UCCCACUCGUUGAUGGACU H, Rt, Rh, M [741-759](18/19) SERPINH1_71 114 ACCGUGGCUUCAUGGUGAA 181 UUCACCAUGAAGCCACGGUH, Rt, Rh, M [891-909] (18/19) SERPINH1_74 115 GAAGGCUGUUGCCAUCUCA 182UGAGAUGGCAACAGCCUUC H, Rt, [1126-1144] (18/19) SERPINH1_75 116GAAGAUGCAGAAGAAGGCA 183 UGCCUUCUUCUGCAUCUUC H, Rt, Rh, [1114-1132] Rb(18/19) SERPINH1_77 117 UGAUGAUGCACCGGACAGA 184 UCUGUCCGGUGCAUCAUCAH, Rh, [933-951] (18/19) SERPINH1_78 118 CCCUUUGACCAGGACAUCA 185UGAUGUCCUGGUCAAAGGG H, Rt, Rh, [1322-1340] (18/19) SERPINH1_80 119CAGUCCAUCAACGAGUGGA 186 UCCACUCGUUGAUGGACUG H, Rt, Rh, M [740-758](18/19) SERPINH1_82 120 CAACCGUGGCUUCAUGGUA 187 UACCAUGAAGCCACGGUUGH, Rt, Rh, M [889-907] (18/19) SERPINH1_83 121 CGACAAGCGCAGCGCGCUA 188UAGCGCGCUGCGCUUGUCG H [721-739] (18/19) SERPINH1_84 122GCAGUCCAUCAACGAGUGA 189 UCACUCGUUGAUGGACUGC H, Rt, Rh, M [739-757](18/19) SERPINH1_86 123 ACAGGCCUCUACAACUACA 190 UGUAGUUGUAGAGGCCUGUH, Rt, Rh, [947-965] Rb, P, D (18/19) SERPINH1_87 124AAGAUGCAGAAGAAGGCUA 191 UAGCCUUCUUCUGCAUCUU H, Rt, Rh, M [1115-1133](18/19) SERPINH1_89 125 CAGCGCGCUGCAGUCCAUA 192 UAUGGACUGCAGCGCGCUGH, Rt, Rh, [730-748] (18/19) SERPINH1_90 126 GCGCAGCGCGCUGCAGUCA 193UGACUGCAGCGCGCUGCGC H, Rt, Rh, [727-745] (18/19)

TABLE 5 SEQ SEQ ID ID Ident NO NO Human gi Name SEN Sense (5′ > 3′) ASAntisense (5′ > 3′) Species Lg 32454740 SERPINH1_1 194GGACAGGCCUCUACAACUA 219 UAGUUGUAGAGGCCUGUCC H, Rt, Rh, 19 [945-963] Rb,P (19/19) SERPINH1_4 195 GAGACACAUGGGUGCUAUU 220 AAUAGCACCCAUGUGUCUC H,Rt, Rh, 19 [1533-1551] M, D (19/19) SERPINH1_12 196 ACAAGAUGCGAGACGAGUU221 AACUCGUCUCGCAUCUUGU H, Rt, Rh, 19 [1464-1482] (19/19) SERPINH1_18197 CCUUUGACCAGGACAUCUA 222 UAGAUGUCCUGGUCAAAGG H, Rt, Rh, 19[1323-1341] (19/19) SERPINH1_29 198 GACCCAUGACCUGCAGAAA 223UUUCUGCAGGUCAUGGGUC H, Rt, Rh, M 19 [1168-1186] (19/19) SERPINH1_30 199CGGACAGGCCUCUACAACU 224 AGUUGUAGAGGCCUGUCCG H, Rt, Rh, 19 [944-962] Rb,P (19/19) SERPINH1_40 200 ACCGGACAGGCCUCUACAA 225 UUGUAGAGGCCUGUCCGGU H,Rt, Rh, 19 [942-960] Rb, P, (19/19) SERPINH1_46 201 GCAGCGCGCUGCAGUCCAU226 AUGGACUGCAGCGCGCUGC H, Rt, Rh, 19 [729-747] (19/19) SERPINH1_47 202GCGCGCUGCAGUCCAUCAA 227 UUGAUGGACUGCAGCGCGC H, Rt, Rh, 19 [732-750](19/19) SERPINH1_50 203 CUGAGACACAUGGGUGCUA 228 UAGCACCCAUGUGUCUCAG H,Rt, Rh, 19 [1531-1549] M, D (19/19) SERPINH1_54 204 AGAAGAAGGCUGUUGCCAU229 AUGGCAACAGCCUUCUUCU H, Rt 19 [1122-1140] (19/19) SERPINH1_55 205AGCUCUCCAGCCUCAUCAU 230 AUGAUGAGGCUGGAGAGCU H, Rt, D, 19 [1017-1035] M,P, Rh (19/19) SERPINH1_56 206 CUGCAGUCCAUCAACGAGU 231ACUCGUUGAUGGACUGCAG H, Rt, Rh, M 19 [737-755] (19/19) SERPINH1_57 207CGCUGCAGUCCAUCAACGA 232 UCGUUGAUGGACUGCAGCG H, Rt, Rh, 19 [735-753](19/19) SERPINH1_58 208 GACAAGAUGCGAGACGAGU 233 ACUCGUCUCGCAUCUUGUC H,Rt, Rh, 19 [1463-1481] (19/19) SERPINH1_63 209 GGGCCUGACUGAGGCCAUU 234AAUGGCCUCAGUCAGGCCC H, Rt 19 [1201-1219] (19/19) SERPINH1_67 210GAUGCAGAAGAAGGCUGUU 235 AACAGCCUUCUUCUGCAUC H, Rt, Rh, M 19 [1117-1135](19/19) SERPINH1_72 211 CACCGGACAGGCCUCUACA 236 UGUAGAGGCCUGUCCGGUG H,Rt, Rh, 19 [941-959] Rb, P (19/19) SERPINH1_73 212 AGAUGCAGAAGAAGGCUGU237 ACAGCCUUCUUCUGCAUCU H, Rt, Rh M 19 [1116-1134] (19/19) SERPINH1_76213 AGCGCGCUGCAGUCCAUCA 238 UGAUGGACUGCAGCGCGCU H, Rt, Rh 19 [731-749](19/19) SERPINH1_79 214 GGAAGAUGCAGAAGAAGGC 239 GCCUUCUUCUGCAUCUUCC H,Rt, Rh, 19 [1113-1131] Rb (19/19) SERPINH1_81 215 GAAGAAGGCUGUUGCCAUC240 GAUGGCAACAGCCUUCUUC H, Rt 19 [1123-1141] (19/19) SERPINH1_85 216UGCAGUCCAUCAACGAGUG 241 CACUCGUUGAUGGACUGCA H, Rt, Rh, M 19 [738-756](19/19) SERPINH1_88 217 CCUGAGACACAUGGGUGCU 242 AGCACCCAUGUGUCUCAGG H,Rt, D, M 19 [1530-1548] (19/19) SERPINH1_91 218 CGCAGCGCGCUGCAGUCCA 243UGGACUGCAGCGCGCUGCG H, Rt, Rh, 19 [728-746] (19/19)

TABLE B Additional Active 19-mer SERPINH1 siRNAs human- Other 32454740No SEQ ID SEN Sense siRNA SEQ ID AS AntiSense siRNA Species ORF:230-1486 1 244 GGCAGACUCUGGUCAAGAA 460 UUCUUGACCAGAGUCUGCC Rh[2009-2027] 3′UTR 2 245 CAGUGAGGCGGAUUGAGAA 461 UUCUCAAUCCGCCUCACUG[1967-1985] 3′UTR 3 246 AGCCUUUGUUGCUAUCAAU 462 AUUGAUAGCAACAAAGGCU Rh[2117-2135] 3′UTR 4 247 CCAUGUUCUUCAAGCCACA 463 UGUGGCUUGAAGAACAUGG Rh,Rb, D [837-855] ORF 5 248 CCCUCUUCUGACACUAAAA 464 UUUUAGUGUCAGAAGAGGG[1850-1868] 3′UTR 6 249 CCUCAAUCAGUAUUCAUAU 465 AUAUGAAUACUGAUUGAGG[1774-1792] 3′UTR 7 250 GAGACACAUGGGUGCUAUU 466 AAUAGCACCCAUGUGUCUC Rh,D, Rt, M [1533-1551] 3′UTR 8 251 GUGACAAGAUGCGAGACGA 467UCGUCUCGCAUCUUGUCAC Rh [1461-1479] ORF 9 252 GCCACACUGGGAUGAGAAA 468UUUCUCAUCCCAGUGUGGC Rh, Rb, M [850-868] ORF 10 253 AGAUGCGAGACGAGUUAUA469 UAUAACUCGUCUCGCAUCU Rh [1467-1485] ORF 11 254 ACGACGACGAGAAGGAAAA470 UUUUCCUUCUCGUCGUCGU [966-984] ORF 12 255 GCCUCUACAACUACUACGA 471UCGUAGUAGUUGUAGAGGC Rb, D [951-969] ORF 13 256 AGAUCAACUUCCGCGACAA 472UUGUCGCGGAAGUUGAUCU D [708-726] ORF 14 257 ACUACUACGACGACGAGAA 473UUCUCGUCGUCGUAGUAGU Rb [960-978] ORF 15 258 AGCCCUCUUCUGACACUAA 474UUAGUGUCAGAAGAGGGCU [1848-1866] 3′UTR 16 259 ACAAGAUGCGAGACGAGUU 475AACUCGUCUCGCAUCUUGU Rh, Rt [1464-1482] ORF 17 260 AGCCACACUGGGAUGAGAA476 UUCUCAUCCCAGUGUGGCU Rh, Rb, M [849-867] ORF 18 261AGGACCAGGCAGUGGAGAA 477 UUCUCCACUGCCUGGUCCU Rh [408-426] ORF 19 262CAGGCAAGAAGGACCUGUA 478 UACAGGUCCUUCUUGCCUG Rh, D [1251-1269] ORF 20 263ACCUGUGAGACCAAAUUGA 479 UCAAUUUGGUCUCACAGGU Rh [1813-1831] 3′UTR 21 264CUUUGUUGCUAUCAAUCCA 480 UGGAUUGAUAGCAACAAAG Rh [2120-2138] 3′UTR 22 265GUGAGACCAAAUUGAGCUA 481 UAGCUCAAUUUGGUCUCAC Rh [1817-1835] 3′UTR 23 266CCCUGAAAGUCCCAGAUCA 482 UGAUCUGGGACUUUCAGGG [1749-1767] 3′UTR 24 267CCUUUGACCAGGACAUCUA 483 UAGAUGUCCUGGUCAAAGG Rh, Rt [1323-1341] ORF 25268 GACCAGGCAGUGGAGAACA 484 UGUUCUCCACUGCCUGGUC Rh [410-428] ORF 26 269CGCGCAACGUGACCUGGAA 485 UUCCAGGUCACGUUGCGCG M [597-615] ORF 27 270AUGAGAAAUUCCACCACAA 486 UUGUGGUGGAAUUUCUCAU Rh [861-879] ORF 28 271GAAGAAACCUGCAGCCGCA 487 UGCGGCUGCAGGUUUCUUC [292-310] ORF 29 272CUCUCGAGCGCCUUGAAAA 488 UUUUCAAGGCGCUCGAGAG [1059-1077] ORF 30 273GGAACAUGAGCCUUUGUUG 489 CAACAAAGGCUCAUGUUCC Rh [2109-2127] 3′UTR 31 274CUCACCUGUGAGACCAAAU 490 AUUUGGUCUCACAGGUGAG Rh [1810-1828] 3′UTR 32 275CUACGACGACGAGAAGGAA 491 UUCCUUCUCGUCGUCGUAG Rb [964-982] ORF 33 276ACCACAAGAUGGUGGACAA 492 UUGUCCACCAUCUUGUGGU Rh, Rb, M, P [873-891] ORF34 277 CUGGCACUGCGGAGAAGUU 493 AACUUCUCCGCAGUGCCAG [318-336] ORF 35 278GGUCCUAUACCGUGGGUGU 494 ACACCCACGGUAUAGGACC Rh [912-930] ORF 36 279CCAACCUCUCCCAACUAUA 495 UAUAGUUGGGAGAGGUUGG Rh [1896-1914] 3′UTR 37 280GAGAAGGAAAAGCUGCAAA 496 UUUGCAGCUUUUCCUUCUC Rh [974-992] ORF 38 281GCCUCUCGAGCGCCUUGAA 497 UUCAAGGCGCUCGAGAGGC [1057-1075] ORF 39 282AGGCCAUUGACAAGAACAA 498 UUGUUCUUGUCAAUGGCCU Rh, D [1212-1230] ORF 40 283GACCCAUGACCUGCAGAAA 499 UUUCUGCAGGUCAUGGGUC Rh, Rt, M [1168-1186] ORF 41284 CUCCUGGCACUGCGGAGAA 500 UUCUCCGCAGUGCCAGGAG [315-333] ORF 42 285CGGACAGGCCUCUACAACU 501 AGUUGUAGAGGCCUGUCCG Rh, Rb, Rt, P [944-962] ORF43 286 GAUGAGAAAUUCCACCACA 502 UGUGGUGGAAUUUCUCAUC Rh [860-878] ORF 44287 CACGCAUGUCAGGCAAGAA 503 UUCUUGCCUGACAUGCGUG Rh, D [1242-1260] ORF 45288 ACCUCUCCCAACUAUAAAA 504 UUUUAUAGUUGGGAGAGGU Rh [1899-1917] 3′UTR 46289 ACCAGGCAGUGGAGAACAU 505 AUGUUCUCCACUGCCUGGU Rh [411-429] ORF 47 290GGGAACAUGAGCCUUUGUU 506 AACAAAGGCUCAUGUUCCC Rh [2108-2126] 3′UTR 48 291AGAAUUCACUCCACUUGGA 507 UCCAAGUGGAGUGAAUUCU Rh [1653-1671] 3′UTR 49 292GGGCAGACUCUGGUCAAGA 508 UCUUGACCAGAGUCUGCCC Rh [2008-2026] 3′UTR 50 293AGAAGGAAAAGCUGCAAAU 509 AUUUGCAGCUUUUCCUUCU Rh [975-993] ORF 51 294GGCAGUGGAGAACAUCCUG 510 CAGGAUGUUCUCCACUGCC Rh [415-433] ORF 52 295GGGAUGAGAAAUUCCACCA 511 UGGUGGAAUUUCUCAUCCC Rh [858-876] ORF 53 296CCAAGCUGUUCUACGCCGA 512 UCGGCGUAGAACAGCUUGG Rh [1365-1383] ORF 54 297ACCGGACAGGCCUCUACAA 513 UUGUAGAGGCCUGUCCGGU Rh, Rb, Rt, P [942-960] ORF55 298 CUGCCUCAAUCAGUAUUCA 514 UGAAUACUGAUUGAGGCAG [1771-1789] 3′UTR 56299 CAGCCCUCUUCUGACACUA 515 UAGUGUCAGAAGAGGGCUG [1847-1865] 3′UTR 57 300CCAGCCUCAUCAUCCUCAU 516 AUGAGGAUGAUGAGGCUGG Rh, D, Rt, M [1023-1041] ORF58 301 AGGGUGACAAGAUGCGAGA 517 UCUCGCAUCUUGUCACCCU Rh, D [1458-1476] ORF59 302 GGACCAGGCAGUGGAGAAC 518 GUUCUCCACUGCCUGGUCC Rh [409-427] ORF 60303 GCAGCGCGCUGCAGUCCAU 519 AUGGACUGCAGCGCGCUGC Rh, Rt [729-747] ORF 61304 GCGCGCUGCAGUCCAUCAA 520 UUGAUGGACUGCAGCGCGC Rh, Rt [732-750] ORF 62305 CCAGAUACCAUGAUGCUGA 521 UCAGCAUCAUGGUAUCUGG Rh [1680-1698] 3′UTR 63306 CUAGUGCGGGACACCCAAA 522 UUUGGGUGUCCCGCACUAG [1400-1418] ORF 64 307AGGCAGUGGAGAACAUCCU 523 AGGAUGUUCUCCACUGCCU Rh [414-432] ORF 65 308CUGAGACACAUGGGUGCUA 524 UAGCACCCAUGUGUCUCAG Rh, D, Rt, M [1531-1549]3′UTR 66 309 GAUUGAGAAGGAGCUCCCA 525 UGGGAGCUCCUUCUCAAUC [1977-1995]3′UTR 67 310 CGCAGACCACCGACGGCAA 526 UUGCCGUCGGUGGUCUGCG D, Rt [762-780]ORF 68 311 CCACACUGGGAUGAGAAAU 527 AUUUCUCAUCCCAGUGUGG Rh [851-869] ORF69 312 GCUCAGUGAGCUUCGCUGA 528 UCAGCGAAGCUCACUGAGC [642-660] ORF 70 313CGCCUUUGAGUUGGACACA 529 UGUGUCCAACUCAAAGGCG Rh [1294-1312] ORF 71 314GGGUCAGCCAGCCCUCUUC 530 GAAGAGGGCUGGCUGACCC Rh [1839-1857] 3′UTR 72 315GGGCUUCUGGGCAGACUCU 531 AGAGUCUGCCCAGAAGCCC Rh [2000-2018] 3′UTR 73 316GGUACCUUCUCACCUGUGA 532 UCACAGGUGAGAAGGUACC Rh [1802-1820] 3′UTR 74 317GCCUGCCUCAAUCAGUAUU 533 AAUACUGAUUGAGGCAGGC [1769-1787] 3′UTR 75 318UCUACAACUACUACGACGA 534 UCGUCGUAGUAGUUGUAGA Rb [954-972] ORF 76 319GGGAAGAUGCAGAAGAAGG 535 CCUUCUUCUGCAUCUUCCC Rh, Rb, Rt [1112-1130] ORF77 320 CGAGAAGGAAAAGCUGCAA 536 UUGCAGCUUUUCCUUCUCG Rh [973-991] ORF 78321 AGAAGAAGGCUGUUGCCAU 537 AUGGCAACAGCCUUCUUCU Rt [1122-1140] ORF 79322 CACAAGCUCUCCAGCCUCA 538 UGAGGCUGGAGAGCUUGUG Rh, D, M, P [1013-1031]ORF 80 323 GGGUGACAAGAUGCGAGAC 539 GUCUCGCAUCUUGUCACCC Rh, D [1459-1477]ORF 81 324 UGUUGGAGCGUGGAAAAAA 540 UUUUUUCCACGCUCCAACA [2190-2208] 3′UTR82 325 CUUUGAGUUGGACACAGAU 541 AUCUGUGUCCAACUCAAAG Rh [1297-1315] ORF 83326 AGCUCUCCAGCCUCAUCAU 542 AUGAUGAGGCUGGAGAGCU Rh, D, Rt, M, P[1017-1035] ORF 84 327 AGCUGUUCUACGCCGACCA 543 UGGUCGGCGUAGAACAGCU Rh[1368-1386] ORF 85 328 CUGCAGUCCAUCAACGAGU 544 ACUCGUUGAUGGACUGCAG Rh,Rt, M [737-755] ORF 86 329 UACGACGACGAGAAGGAAA 545 UUUCCUUCUCGUCGUCGUA[965-983] ORF 87 330 CCUAGUGCGGGACACCCAA 546 UUGGGUGUCCCGCACUAGG[1399-1417] ORF 88 331 CUUCUCACCUGUGAGACCA 547 UGGUCUCACAGGUGAGAAG Rh[1807-1825] 3′UTR 89 332 AGUUGGACACAGAUGGCAA 548 UUGCCAUCUGUGUCCAACU[1302-1320] ORF 90 333 CAGUGGAGAACAUCCUGGU 549 ACCAGGAUGUUCUCCACUG Rh[417-435] ORF 91 334 CCAGCUAGAAUUCACUCCA 550 UGGAGUGAAUUCUAGCUGG Rh[1647-1665] 3′UTR 92 335 CGCUGCAGUCCAUCAACGA 551 UCGUUGAUGGACUGCAGCG Rh,Rt [735-753] ORF 93 336 CCAAGGACCAGGCAGUGGA 552 UCCACUGCCUGGUCCUUGG Rh[405-423] ORF 94 337 AGUUCUUCAAAGAUAGGGA 553 UCCCUAUCUUUGAAGAACU[2082-2100] 3′UTR 95 338 CGGACCUUCCCAGCUAGAA 554 UUCUAGCUGGGAAGGUCCG Rh[1638-1656] 3′UTR 96 339 GACAAGAUGCGAGACGAGU 555 ACUCGUCUCGCAUCUUGUC Rh,Rt [1463-1481] ORF 97 340 CCAAGAUCAACUUCCGCGA 556 UCGCGGAAGUUGAUCUUGG D[705-723] ORF 98 341 CCCAUCACGUGGAGCCUCU 557 AGAGGCUCCACGUGAUGGG Rh[1044-1062] ORF 99 342 CCAUGAUGCUGAGCCCGGA 558 UCCGGGCUCAGCAUCAUGG[1687-1705] 3′UTR 100 343 AGCCUGCCUCAAUCAGUAU 559 AUACUGAUUGAGGCAGGCU[1768-1786] 3′UTR 101 344 CGGCCUAAGGGUGACAAGA 560 UCUUGUCACCCUUAGGCCG Rh[1451-1469] ORF 102 345 GGGCCUGACUGAGGCCAUU 561 AAUGGCCUCAGUCAGGCCC Rt[1201-1219] ORF 103 346 UCACCUGUGAGACCAAAUU 562 AAUUUGGUCUCACAGGUGA Rh[1811-1829] 3′UTR 104 347 GAGGCCAUUGACAAGAACA 563 UGUUCUUGUCAAUGGCCUCRh, D [1211-1229] ORF 105 348 GCUCCUGGCACUGCGGAGA 564UCUCCGCAGUGCCAGGAGC [314-332] ORF 106 349 GGCGCCUGGUCCGGCCUAA 565UUAGGCCGGACCAGGCGCC Rh [1440-1458] ORF 107 350 CCAGCCCUCUUCUGACACU 566AGUGUCAGAAGAGGGCUGG [1846-1864] 3′UTR 108 351 ACUACGACGACGAGAAGGA 567UCCUUCUCGUCGUCGUAGU Rb [963-981] ORF 109 352 CCUAUACCGUGGGUGUCAU 568AUGACACCCACGGUAUAGG Rh, D, P [915-933] ORF 110 353 GACCCAGCUCAGUGAGCUU569 AAGCUCACUGAGCUGGGUC [636-654] ORF 111 354 UGGGUGUCAUGAUGAUGCA 570UGCAUCAUCAUGACACCCA Rh [924-942] ORF 112 355 CCAAGGGUGUGGUGGAGGU 571ACCUCCACCACACCCUUGG Rh, D [1149-1167] ORF 113 356 AGGUCACCAAGGACGUGGA572 UCCACGUCCUUGGUGACCU Rh, D [789-807] ORF 114 357 CCCUGGCCGCCGAGGUGAA573 UUCACCUCGGCGGCCAGGG [276-294] ORF 115 358 AGCACUCCAAGAUCAACUU 574AAGUUGAUCUUGGAGUGCU Rh, D [699-717] ORF 116 359 CCUGGCACUGCGGAGAAGU 575ACUUCUCCGCAGUGCCAGG [317-335] ORF 117 360 GAUGCAGAAGAAGGCUGUU 576AACAGCCUUCUUCUGCAUC Rh, Rt, M [1117-1135] ORF 118 361CCCACAAGCUCUCCAGCCU 577 AGGCUGGAGAGCUUGUGGG Rh, D, P [1011-1029] ORF 119362 CUCUUCUGACACUAAAACA 578 UGUUUUAGUGUCAGAAGAG [1852-1870] 3′UTR 120363 ACGAGAAGGAAAAGCUGCA 579 UGCAGCUUUUCCUUCUCGU Rh [972-990] ORF 121 364UGAAAAGCUGCUAACCAAA 580 UUUGGUUAGCAGCUUUUCA [1072-1090] ORF 122 365UCUCACCUGUGAGACCAAA 581 UUUGGUCUCACAGGUGAGA Rh [1809-1827] 3′UTR 123 366CAUGAUGAUGCACCGGACA 582 UGUCCGGUGCAUCAUCAUG Rh [931-949] ORF 124 367GGAUUGAGAAGGAGCUCCC 583 GGGAGCUCCUUCUCAAUCC [1976-1994] 3′UTR 125 368CCUUCAUCUUCCUAGUGCG 584 CGCACUAGGAAGAUGAAGG [1389-1407] ORF 126 369GGCCUGGCCUUCAGCUUGU 585 ACAAGCUGAAGGCCAGGCC [374-392] ORF 127 370GGUCAGCCAGCCCUCUUCU 586 AGAAGAGGGCUGGCUGACC Rh [1840-1858] 3′UTR 128 371UUCUCACCUGUGAGACCAA 587 UUGGUCUCACAGGUGAGAA Rh [1808-1826] 3′UTR 129 372CGCAGCAGCUCCUGGCACU 588 AGUGCCAGGAGCUGCUGCG [307-325] ORF 130 373GCCAUGUUCUUCAAGCCAC 589 GUGGCUUGAAGAACAUGGC Rh, Rb, D [836-854] ORF 131374 AGGCAGUGCUGAGCGCCGA 590 UCGGCGCUCAGCACUGCCU [510-528] ORF 132 375CACCUGUGAGACCAAAUUG 591 CAAUUUGGUCUCACAGGUG Rh [1812-1830] 3′UTR 133 376CACCGGACAGGCCUCUACA 592 UGUAGAGGCCUGUCCGGUG Rh, Rb, Rt, P [941-959] ORF134 377 AGCUAGAAUUCACUCCACU 593 AGUGGAGUGAAUUCUAGCU Rh [1649-1667] 3′UTR135 378 AGAUGCAGAAGAAGGCUGU 594 ACAGCCUUCUUCUGCAUCU Rh, Rt, M[1116-1134] ORF 136 379 CCCUGCUAGUCAACGCCAU 595 AUGGCGUUGACUAGCAGGG Rh[822-840] ORF 137 380 ACAACUACUACGACGACGA 596 UCGUCGUCGUAGUAGUUGU Rb[957-975] ORF 138 381 GCUCCUGAGACACAUGGGU 597 ACCCAUGUGUCUCAGGAGC D[1527-1545] 3′UTR 139 382 UGGAGAACAUCCUGGUGUC 598 GACACCAGGAUGUUCUCCA Rh[420-438] ORF 140 383 AGCGCGCUGCAGUCCAUCA 599 UGAUGGACUGCAGCGCGCU Rh, Rt[731-749] ORF 141 384 CGCCUUGAAAAGCUGCUAA 600 UUAGCAGCUUUUCAAGGCG[1067-1085] ORF 142 385 GCCUUUGUUGCUAUCAAUC 601 GAUUGAUAGCAACAAAGGC Rh[2118-2136] 3′UTR 143 386 CUCUACAACUACUACGACG 602 CGUCGUAGUAGUUGUAGAG Rb[953-971] ORF 144 387 CGCUCACUCAGCAACUCCA 603 UGGAGUUGCUGAGUGAGCG Rh[575-593] ORF 145 388 GGUACCAGCCUUGGAUACU 604 AGUAUCCAAGGCUGGUACC Rh[1571-1589] 3′UTR 146 389 GCCUGACUGAGGCCAUUGA 605 UCAAUGGCCUCAGUCAGGC Rh[1203-1221] ORF 147 390 UGAGCUUCGCUGAUGACUU 606 AAGUCAUCAGCGAAGCUCA Rh[648-666] ORF 148 391 CCAGCCUUGGAUACUCCAU 607 AUGGAGUAUCCAAGGCUGG Rh[1575-1593] 3′UTR 149 392 AAAGGCUCCUGAGACACAU 608 AUGUGUCUCAGGAGCCUUU[1523-1541] 3′UTR 150 393 UGACCCAUGACCUGCAGAA 609 UUCUGCAGGUCAUGGGUCARh, Rt, M [1167-1185] ORF 151 394 CCUGUGAGACCAAAUUGAG 610CUCAAUUUGGUCUCACAGG Rh [1814-1832] 3′UTR 152 395 GCGGACCUUCCCAGCUAGA 611UCUAGCUGGGAAGGUCCGC Rh [1637-1655] 3′UTR 153 396 GGAAGAUGCAGAAGAAGGC 612GCCUUCUUCUGCAUCUUCC Rh, Rb, Rt [1113-1131] ORF 154 397UGCCCAAGGGUGUGGUGGA 613 UCCACCACACCCUUGGGCA Rh, D [1146-1164] ORF 155398 GGAGCCUCUCGAGCGCCUU 614 AAGGCGCUCGAGAGGCUCC [1054-1072] ORF 156 399GACUCUGGUCAAGAAGCAU 615 AUGCUUCUUGACCAGAGUC Rh [2013-2031] 3′UTR 157 400CAGGCAGUGGAGAACAUCC 616 GGAUGUUCUCCACUGCCUG Rh [413-431] ORF 158 401CAAGCCUGCCUCAAUCAGU 617 ACUGAUUGAGGCAGGCUUG Rh [1766-1784] 3′UTR 159 402CUGGAAGCUGGGCAGCCGA 618 UCGGCUGCCCAGCUUCCAG [610-628] ORF 160 403GAAGAAGGCUGUUGCCAUC 619 GAUGGCAACAGCCUUCUUC Rt [1123-1141] ORF 161 404GGGCGAGCUGCUGCGCUCA 620 UGAGCGCAGCAGCUCGCCC Rh [562-580] ORF 162 405AAGCCACACUGGGAUGAGA 621 UCUCAUCCCAGUGUGGCUU Rh, Rb, M [848-866] ORF 163406 GUGUGGUGGAGGUGACCCA 622 UGGGUCACCUCCACCACAC Rh, D [1155-1173] ORF164 407 CCGCCUUUGAGUUGGACAC 623 GUGUCCAACUCAAAGGCGG Rh [1293-1311] ORF165 408 GGCCAUUGACAAGAACAAG 624 CUUGUUCUUGUCAAUGGCC Rh, D [1213-1231]ORF 166 409 UGCCUCAAUCAGUAUUCAU 625 AUGAAUACUGAUUGAGGCA [1772-1790]3′UTR 167 410 CCUUCCCAGCUAGAAUUCA 626 UGAAUUCUAGCUGGGAAGG Rh [1642-1660]3′UTR 168 411 GGGACCUGGGCCAUAGUCA 627 UGACUAUGGCCCAGGUCCC [1721-1739]3′UTR 169 412 CGAGGUGAAGAAACCUGCA 628 UGCAGGUUUCUUCACCUCG Rh [286-304]ORF 170 413 GCCUUUGAGUUGGACACAG 629 CUGUGUCCAACUCAAAGGC Rh [1295-1313]ORF 171 414 AGCGGACCUUCCCAGCUAG 630 CUAGCUGGGAAGGUCCGCU Rh [1636-1654]3′UTR 172 415 CGCAUGUCAGGCAAGAAGG 631 CCUUCUUGCCUGACAUGCG Rh, D[1244-1262] ORF 173 416 ACAACUGCGAGCACUCCAA 632 UUGGAGUGCUCGCAGUUGU Rh,D [690-708] ORF 174 417 GAGGCGGAUUGAGAAGGAG 633 CUCCUUCUCAAUCCGCCUC[1971-1989] 3′UTR 175 418 GGCCGCCGAGGUGAAGAAA 634 UUUCUUCACCUCGGCGGCC[280-298] ORF 176 419 CAGCUCUAUCCCAACCUCU 635 AGAGGUUGGGAUAGAGCUG[1886-1904] 3′UTR 177 420 AGCUGGGCAGCCGACUGUA 636 UACAGUCGGCUGCCCAGCU[615-633] ORF 178 421 GCCAUUGACAAGAACAAGG 637 CCUUGUUCUUGUCAAUGGC Rh, D[1214-1232] ORF 179 422 CGCCAUGUUCUUCAAGCCA 638 UGGCUUGAAGAACAUGGCG Rh,Rb, P [835-853] ORF 180 423 CCGAGGUCACCAAGGACGU 639 ACGUCCUUGGUGACCUCGGRh, D [786-804] ORF 181 424 GGACCCAGCUCAGUGAGCU 640 AGCUCACUGAGCUGGGUCC[635-653] ORF 182 425 CCAAUGACAUUUUGUUGGA 641 UCCAACAAAAUGUCAUUGG[2178-2196] 3′UTR 183 426 AGUGAGGCGGAUUGAGAAG 642 CUUCUCAAUCCGCCUCACU[1968-1986] 3′UTR 184 427 UGCAGUCCAUCAACGAGUG 643 CACUCGUUGAUGGACUGCARh, Rt, M [738-756] ORF 185 428 UGUCACGCAUGUCAGGCAA 644UUGCCUGACAUGCGUGACA Rh, D [1239-1257] ORF 186 429 CGACGACGAGAAGGAAAAG645 CUUUUCCUUCUCGUCGUCG [967-985] ORF 187 430 ACAAGAACAAGGCCGACUU 646AAGUCGGCCUUGUUCUUGU Rh [1221-1239] ORF 188 431 CUUCAAGCCACACUGGGAU 647AUCCCAGUGUGGCUUGAAG Rh, Rb, D [844-862] ORF 189 432 CCUGGGCCAUAGUCAUUCU648 AGAAUGACUAUGGCCCAGG [1725-1743] 3′UTR 190 433 UUUGUUGGAGCGUGGAAAA649 UUUUCCACGCUCCAACAAA [2188-2206] 3′UTR 191 434 AGAACAUCCUGGUGUCACC650 GGUGACACCAGGAUGUUCU [423-441] ORF 192 435 ACGCCACCGCCUUUGAGUU 651AACUCAAAGGCGGUGGCGU Rh [1287-1305] ORF 193 436 GUGAGGUACCAGCCUUGGA 652UCCAAGGCUGGUACCUCAC Rh [1567-1585] 3′UTR 194 437 GCGCCUUCUGCCUCCUGGA 653UCCAGGAGGCAGAAGGCGC [252-270] ORF 195 438 GCCUGGCCUUCAGCUUGUA 654UACAAGCUGAAGGCCAGGC [375-393] ORF 196 439 CCCGGAAACUCCACAUCCU 655AGGAUGUGGAGUUUCCGGG [1700-1718] 3′UTR 197 440 UCUUCAAGCCACACUGGGA 656UCCCAGUGUGGCUUGAAGA Rh, Rb, D [843-861] ORF 198 441 UGUUGCUAUCAAUCCAAGA657 UCUUGGAUUGAUAGCAACA Rh [2123-2141] 3′UTR 199 442 GAGUGGGCCGCGCAGACCA658 UGGUCUGCGCGGCCCACUC [752-770] ORF 200 443 CCUGAGACACAUGGGUGCU 659AGCACCCAUGUGUCUCAGG D, Rt, M [1530-1548] 3′UTR 201 444AGCCGACUGUACGGACCCA 660 UGGGUCCGUACAGUCGGCU [623-641] ORF 202 445GGGCCUCAGGGUGCACACA 661 UGUGUGCACCCUGAGGCCC [1486-1504] 3′UTR 203 446ACUGGGAUGAGAAAUUCCA 662 UGGAAUUUCUCAUCCCAGU Rh [855-873] ORF 204 447AGAAUGACCUGGCCGCAGU 663 ACUGCGGCCAGGUCAUUCU [1952-1970] 3′UTR 205 448CAUAUUUAUAGCCAGGUAC 664 GUACCUGGCUAUAAAUAUG Rh [1788-1806] 3′UTR 206 449AGGUGACCCAUGACCUGCA 665 UGCAGGUCAUGGGUCACCU Rh, Rt, M [1164-1182] ORF207 450 GCGCUGCAGUCCAUCAACG 666 CGUUGAUGGACUGCAGCGC Rh, Rt [734-752] ORF208 451 GGUGACAAGAUGCGAGACG 667 CGUCUCGCAUCUUGUCACC Rh [1460-1478] ORF209 452 CUUCAAAGAUAGGGAGGGA 668 UCCCUCCCUAUCUUUGAAG [2086-2104] 3′UTR210 453 AGCUGCAAAUCGUGGAGAU 669 AUCUCCACGAUUUGCAGCU Rh [984-1002] ORF211 454 GUGGAGAACAUCCUGGUGU 670 ACACCAGGAUGUUCUCCAC Rh [419-437] ORF 212455 GAACAAGGCCGACUUGUCA 671 UGACAAGUCGGCCUUGUUC Rh [1225-1243] ORF 213456 CAUGAUGCUGAGCCCGGAA 672 UUCCGGGCUCAGCAUCAUG [1688-1706] 3′UTR 214457 GCGCCUUGAAAAGCUGCUA 673 UAGCAGCUUUUCAAGGCGC Rh [1066-1084] ORF 215458 GCAGACUCUGGUCAAGAAG 674 CUUCUUGACCAGAGUCUGC Rh [2010-2028] 3′UTR 216459 CCAGGCAGUGGAGAACAUC 675 GAUGUUCUCCACUGCCUGG Rh [412-430] ORF

TABLE C Cross-Species 19-mer SERPINH1 siRNAs human- 32454740 No. SEQ IDSEN Sense siRNA SEQ ID AS AntiSense siRNA Other Species ORF: 230-1486 1676 CACUACAACUGCGAGCACU 973 AGUGCUCGCAGUUGUAGUG Rh, D [686-704] ORF 2677 AACCGUGGCUUCAUGGUGA 974 UCACCAUGAAGCCACGGUU Rh, Rt, M [890-908] ORF3 678 GGCAAGAAGGACCUGUACC 975 GGUACAGGUCCUUCUUGCC Rh, D, M [1253-1271]ORF 4 679 GGUGGACAACCGUGGCUUC 976 GAAGCCACGGUUGUCCACC Rh, M [883-901]ORF 5 680 AGGCCAUGGCCAAGGACCA 977 UGGUCCUUGGCCAUGGCCU Rh, D [396-414]ORF 6 681 CGCAGCGCGCUGCAGUCCA 978 UGGACUGCAGCGCGCUGCG Rh, Rt [728-746]ORF 7 682 AGCAGCAAGCAGCACUACA 979 UGUAGUGCUGCUUGCUGCU Rh, D [674-692]ORF 8 683 GGCCUCUACAACUACUACG 980 CGUAGUAGUUGUAGAGGCC Rb, D [950-968]ORF 9 684 GAAGAUGCAGAAGAAGGCU 981 AGCCUUCUUCUGCAUCUUC Rh, Rb, Rt[1114-1132] ORF 10 685 GGCUCCUGAGACACAUGGG 982 CCCAUGUGUCUCAGGAGCC D[1526-1544] 3′UTR 11 686 AGCAAGCAGCACUACAACU 983 AGUUGUAGUGCUGCUUGCU Rh,D [677-695] ORF 12 687 GGAGGUGACCCAUGACCUG 984 CAGGUCAUGGGUCACCUCC Rh,Rt, M [1162-1180] ORF 13 688 CCCUUUGACCAGGACAUCU 985 AGAUGUCCUGGUCAAAGGGRh, Rt [1322-1340] ORF 14 689 CUCCUGAGACACAUGGGUG 986CACCCAUGUGUCUCAGGAG D [1528-1546] 3′UTR 15 690 AAGGCUCCUGAGACACAUG 987CAUGUGUCUCAGGAGCCUU D [1524-1542] 3′UTR 16 691 CGCGCUGCAGUCCAUCAAC 988GUUGAUGGACUGCAGCGCG Rh, Rt [733-751] ORF 17 692 AGGGUGUGGUGGAGGUGAC 989GUCACCUCCACCACACCCU Rh, D [1152-1170] ORF 18 693 AGCACUACAACUGCGAGCA 990UGCUCGCAGUUGUAGUGCU Rh, D [684-702] ORF 19 694 GGCUCCCUGCUAUUCAUUG 991CAAUGAAUAGCAGGGAGCC D [1421-1439] ORF 20 695 GCGCGCAACGUGACCUGGA 992UCCAGGUCACGUUGCGCGC M [596-614] ORF 21 696 GCUGCAGUCCAUCAACGAG 993CUCGUUGAUGGACUGCAGC Rh, Rt [736-754] ORF 22 697 ACCAAAGAGCAGCUGAAGA 994UCUUCAGCUGCUCUUUGGU Rh, Rb, P [1085-1103] ORF 23 698 CCAAGGACGUGGAGCGCAC995 GUGCGCUCCACGUCCUUGG Rh, D [795-813] ORF 24 699 UGUUCUUCAAGCCACACUG996 CAGUGUGGCUUGAAGAACA Rh, Rb, D [840-858] ORF 25 700GCCCAAGGGUGUGGUGGAG 997 CUCCACCACACCCUUGGGC Rh, D [1147-1165] ORF 26 701ACAGGCCUCUACAACUACU 998 AGUAGUUGUAGAGGCCUGU Rh, Rb, D, Rt, P [947-965]ORF 27 702 UGCGCAGCAGCAAGCAGCA 999 UGCUGCUUGCUGCUGCGCA Rh, D [669-687]ORF 28 703 GGUGGAGGUGACCCAUGAC 1000 GUCAUGGGUCACCUCCACC Rh, Rt, M[1159-1177] ORF 29 704 CUUUGACCAGGACAUCUAC 1001 GUAGAUGUCCUGGUCAAAG Rh,Rt [1324-1342] ORF 30 705 AAGGGUGUGGUGGAGGUGA 1002 UCACCUCCACCACACCCUURh, D [1151-1169] ORF 31 706 UCCUAUACCGUGGGUGUCA 1003UGACACCCACGGUAUAGGA Rh, D, P [914-932] ORF 32 707 GCGCAGACCACCGACGGCA1004 UGCCGUCGGUGGUCUGCGC D [761-779] ORF 33 708 CGCAGCAGCAAGCAGCACU 1005AGUGCUGCUUGCUGCUGCG Rh, D [671-689] ORF 34 709 GCCUCAUCAUCCUCAUGCC 1006GGCAUGAGGAUGAUGAGGC Rh, D, Rt, M [1026-1044] ORF 35 710UCUCCAGCCUCAUCAUCCU 1007 AGGAUGAUGAGGCUGGAGA Rh, D, Rt, M [1020-1038]ORF 36 711 CCAUUGACAAGAACAAGGC 1008 GCCUUGUUCUUGUCAAUGG Rh, D[1215-1233] ORF 37 712 AGCAGCACUACAACUGCGA 1009 UCGCAGUUGUAGUGCUGCU Rh,D [681-699] ORF 38 713 UGCACCGGACAGGCCUCUA 1010 UAGAGGCCUGUCCGGUGCA Rh,Rb, Rt, P [939-957] ORF 39 714 ACUCCAAGAUCAACUUCCG 1011CGGAAGUUGAUCUUGGAGU Rh, D, Rt, M [702-720] ORF 40 715UGGACAACCGUGGCUUCAU 1012 AUGAAGCCACGGUUGUCCA Rh, M [885-903] ORF 41 716GAGCAGCUGAAGAUCUGGA 1013 UCCAGAUCUUCAGCUGCUC Rh, D [1091-1109] ORF 42717 CAGAAGAAGGCUGUUGCCA 1014 UGGCAACAGCCUUCUUCUG Rt [1121-1139] ORF 43718 AGGCAAGAAGGACCUGUAC 1015 GUACAGGUCCUUCUUGCCU Rh, D [1252-1270] ORF44 719 CCUCUACAACUACUACGAC 1016 GUCGUAGUAGUUGUAGAGG Rb, D [952-970] ORF45 720 AGCAGCUGAAGAUCUGGAU 1017 AUCCAGAUCUUCAGCUGCU Rh, D [1092-1110]ORF 46 721 AACUACUACGACGACGAGA 1018 UCUCGUCGUCGUAGUAGUU Rb [959-977] ORF47 722 GGCAAGCUGCCCGAGGUCA 1019 UGACCUCGGGCAGCUUGCC Rh, D [776-794] ORF48 723 CCGGACAGGCCUCUACAAC 1020 GUUGUAGAGGCCUGUCCGG Rh, Rb, Rt, P[943-961] ORF 49 724 GCUCCCUGCUAUUCAUUGG 1021 CCAAUGAAUAGCAGGGAGC D[1422-1440] ORF 50 725 AACUGCGAGCACUCCAAGA 1022 UCUUGGAGUGCUCGCAGUU Rh,D [692-710] ORF 51 726 GACACAUGGGUGCUAUUGG 1023 CCAAUAGCACCCAUGUGUC Rh,Rt, M [1535-1553] 3′UTR 52 727 GCACCGGACAGGCCUCUAC 1024GUAGAGGCCUGUCCGGUGC Rh, Rb, Rt, P [940-958] ORF 53 728AGCGCAGCGCGCUGCAGUC 1025 GACUGCAGCGCGCUGCGCU Rh, Rt [726-744] ORF 54 729GGACGUGGAGCGCACGGAC 1026 GUCCGUGCGCUCCACGUCC Rh, D [799-817] ORF 55 730CAGCCUCAUCAUCCUCAUG 1027 CAUGAGGAUGAUGAGGCUG Rh, D, Rt, M [1024-1042]ORF 56 731 AAGAUCAACUUCCGCGACA 1028 UGUCGCGGAAGUUGAUCUU D [707-725] ORF57 732 GCGCAACGUGACCUGGAAG 1029 CUUCCAGGUCACGUUGCGC M [598-616] ORF 58733 ACUGCGAGCACUCCAAGAU 1030 AUCUUGGAGUGCUCGCAGU Rh, D [693-711] ORF 59734 GUGGACAACCGUGGCUUCA 1031 UGAAGCCACGGUUGUCCAC Rh, M [884-902] ORF 60735 CCACAAGCUCUCCAGCCUC 1032 GAGGCUGGAGAGCUUGUGG Rh, D, P [1012-1030]ORF 61 736 CAAGAUGGUGGACAACCGU 1033 ACGGUUGUCCACCAUCUUG Rh, Rb, M, P[877-895] ORF 62 737 CGAGCACUCCAAGAUCAAC 1034 GUUGAUCUUGGAGUGCUCG Rh, D[697-715] ORF 63 738 AGCUGCCCGAGGUCACCAA 1035 UUGGUGACCUCGGGCAGCU Rh, D[780-798] ORF 64 739 GGACAUCUACGGGCGCGAG 1036 CUCGCGCCCGUAGAUGUCC D[1333-1351] ORF 65 740 AGGACAUCUACGGGCGCGA 1037 UCGCGCCCGUAGAUGUCCU D[1332-1350] ORF 66 741 UGUCAGGCAAGAAGGACCU 1038 AGGUCCUUCUUGCCUGACA Rh,D [1248-1266] ORF 67 742 GGGUGUGGUGGAGGUGACC 1039 GGUCACCUCCACCACACCCRh, D [1153-1171] ORF 68 743 CAAGCUCUCCAGCCUCAUC 1040GAUGAGGCUGGAGAGCUUG Rh, D, M, P [1015-1033] ORF 69 744GUGACCCAUGACCUGCAGA 1041 UCUGCAGGUCAUGGGUCAC Rh, Rt, M [1166-1184] ORF70 745 GUUCUUCAAGCCACACUGG 1042 CCAGUGUGGCUUGAAGAAC Rh, Rb, D [841-859]ORF 71 746 ACAUCUACGGGCGCGAGGA 1043 UCCUCGCGCCCGUAGAUGU D, M [1335-1353]ORF 72 747 UGGAGGUGACCCAUGACCU 1044 AGGUCAUGGGUCACCUCCA Rh, Rt, M[1161-1179] ORF 73 748 UGCAGAAGAAGGCUGUUGC 1045 GCAACAGCCUUCUUCUGCA Rt[1119-1137] ORF 74 749 UGUACCAGGCCAUGGCCAA 1046 UUGGCCAUGGCCUGGUACA Rh,D [390-408] ORF 75 750 UGUGGUGGAGGUGACCCAU 1047 AUGGGUCACCUCCACCACA Rh,D [1156-1174] ORF 76 751 AGAAGGACCUGUACCUGGC 1048 GCCAGGUACAGGUCCUUCURh, D [1257-1275] ORF 77 752 AGCAGCUGCGCGACGAGGA 1049UCCUCGUCGCGCAGCUGCU Rh, D [528-546] ORF 78 753 ACGCCAUGUUCUUCAAGCC 1050GGCUUGAAGAACAUGGCGU Rh, Rb, P [834-852] ORF 79 754 ACAAGAUGGUGGACAACCG1051 CGGUUGUCCACCAUCUUGU Rh, Rb, M, P [876-894] ORF 80 755CUGCGAGCACUCCAAGAUC 1052 GAUCUUGGAGUGCUCGCAG Rh, D [694-712] ORF 81 756GUCACGCAUGUCAGGCAAG 1053 CUUGCCUGACAUGCGUGAC Rh, D [1240-1258] ORF 82757 ACGCAUGUCAGGCAAGAAG 1054 CUUCUUGCCUGACAUGCGU Rh, D [1243-1261] ORF83 758 UGCUAUUCAUUGGGCGCCU 1055 AGGCGCCCAAUGAAUAGCA D [1428-1446] ORF 84759 UGCGCGACGAGGAGGUGCA 1056 UGCACCUCCUCGUCGCGCA Rh, D [534-552] ORF 85760 GCAGCUGAAGAUCUGGAUG 1057 CAUCCAGAUCUUCAGCUGC Rh, D [1093-1111] ORF86 761 CCAUGACCUGCAGAAACAC 1058 GUGUUUCUGCAGGUCAUGG Rh, Rt, M[1171-1189] ORF 87 762 AAGCUCUCCAGCCUCAUCA 1059 UGAUGAGGCUGGAGAGCUU Rh,D, Rt, M, P [1016-1034] ORF 88 763 CAGCAAGCAGCACUACAAC 1060GUUGUAGUGCUGCUUGCUG Rh, D [676-694] ORF 89 764 AUGUUCUUCAAGCCACACU 1061AGUGUGGCUUGAAGAACAU Rh, Rb, D [839-857] ORF 90 765 UCCUGAGACACAUGGGUGC1062 GCACCCAUGUGUCUCAGGA D, Rt, M [1529-1547] 3′UTR 91 766CACUCCAAGAUCAACUUCC 1063 GGAAGUUGAUCUUGGAGUG Rh, D, Rt, M [701-719] ORF92 767 AAGGGUGACAAGAUGCGAG 1064 CUCGCAUCUUGUCACCCUU Rh, D [1457-1475]ORF 93 768 GACAGGCCUCUACAACUAC 1065 GUAGUUGUAGAGGCCUGUC Rh, Rb, Rt, P[946-964] ORF 94 769 ACCCAUGACCUGCAGAAAC 1066 GUUUCUGCAGGUCAUGGGU Rh,Rt, M [1169-1187] ORF 95 770 CACCACAAGAUGGUGGACA 1067UGUCCACCAUCUUGUGGUG Rh, Rb, M, P [872-890] ORF 96 771GCAGAAGAAGGCUGUUGCC 1068 GGCAACAGCCUUCUUCUGC Rt [1120-1138] ORF 97 772GUGGUGGAGGUGACCCAUG 1069 CAUGGGUCACCUCCACCAC Rh, Rb, Rt, M [1157-1175]ORF 98 773 AGGCCUCUACAACUACUAC 1070 GUAGUAGUUGUAGAGGCCU Rh, Rb, D, Rt, P[949-967] ORF 99 774 GGUGACCCAUGACCUGCAG 1071 CUGCAGGUCAUGGGUCACC Rh,Rt, M [1165-1183] ORF 100 775 GCCGAGGUGAAGAAACCUG 1072CAGGUUUCUUCACCUCGGC Rh, Rt [284-302] ORF 101 776 CAACUACUACGACGACGAG1073 CUCGUCGUCGUAGUAGUUG Rb [958-976] ORF 102 777 CAAGAAGGACCUGUACCUG1074 CAGGUACAGGUCCUUCUUG Rh, D, M [1255-1273] ORF 103 778UGUUCCACGCCACCGCCUU 1075 AAGGCGGUGGCGUGGAACA D [1281-1299] ORF 104 779CCCUGCUAUUCAUUGGGCG 1076 CGCCCAAUGAAUAGCAGGG D [1425-1443] ORF 105 780CCGUGGCUUCAUGGUGACU 1077 AGUCACCAUGAAGCCACGG Rh, Rt, M [892-910] ORF 106781 CUACAACUACUACGACGAC 1078 GUCGUCGUAGUAGUUGUAG Rb [955-973] ORF 107782 GCAGCACUACAACUGCGAG 1079 CUCGCAGUUGUAGUGCUGC Rh, D [682-700] ORF 108783 UGGUGGACAACCGUGGCUU 1080 AAGCCACGGUUGUCCACCA Rh, M [882-900] ORF 109784 AGACCACCGACGGCAAGCU 1081 AGCUUGCCGUCGGUGGUCU D, Rt [765-783] ORF 110785 AGAAACACCUGGCUGGGCU 1082 AGCCCAGCCAGGUGUUUCU D [1182-1200] ORF 111786 ACCAAGGACGUGGAGCGCA 1083 UGCGCUCCACGUCCUUGGU Rh, D [794-812] ORF 112787 CCGAGGUGAAGAAACCUGC 1084 GCAGGUUUCUUCACCUCGG Rh, Rt [285-303] ORF113 788 ACUACAACUGCGAGCACUC 1085 GAGUGCUCGCAGUUGUAGU Rh, D [687-705] ORF114 789 ACAAGCUCUCCAGCCUCAU 1086 AUGAGGCUGGAGAGCUUGU Rh, D, M, P[1014-1032] ORF 115 790 AGGACGUGGAGCGCACGGA 1087 UCCGUGCGCUCCACGUCCU Rh,D [798-816] ORF 116 791 GCUAUUCAUUGGGCGCCUG 1088 CAGGCGCCCAAUGAAUAGC D[1429-1447] ORF 117 792 AACUUCCGCGACAAGCGCA 1089 UGCGCUUGUCGCGGAAGUU D[713-731] ORF 118 793 GCUCUCCAGCCUCAUCAUC 1090 GAUGAUGAGGCUGGAGAGC Rh,D, Rt, M, P [1018-1036] ORF 119 794 AGAAGGCUGUUGCCAUCUC 1091GAGAUGGCAACAGCCUUCU Rt [1125-1143] ORF 120 795 GGUCACCAAGGACGUGGAG 1092CUCCACGUCCUUGGUGACC Rh, D [790-808] ORF 121 796 AGCUGCGCGACGAGGAGGU 1093ACCUCCUCGUCGCGCAGCU Rh, D [531-549] ORF 122 797 CCCGAGGUCACCAAGGACG 1094CGUCCUUGGUGACCUCGGG Rh, D [785-803] ORF 123 798 AUGUCAGGCAAGAAGGACC 1095GGUCCUUCUUGCCUGACAU Rh, D [1247-1265] ORF 124 799 CGAGGUCACCAAGGACGUG1096 CACGUCCUUGGUGACCUCG Rh, D [787-805] ORF 125 800 GAUGCACCGGACAGGCCUC1097 GAGGCCUGUCCGGUGCAUC Rh, Rb, Rt, M, P [937-955] ORF 126 801GCACUACAACUGCGAGCAC 1098 GUGCUCGCAGUUGUAGUGC Rh, D [685-703] ORF 127 802CCACAAGAUGGUGGACAAC 1099 GUUGUCCACCAUCUUGUGG Rh, Rb, M, P [874-892] ORF128 803 CAAGGGUGUGGUGGAGGUG 1100 CACCUCCACCACACCCUUG Rh, D [1150-1168]ORF 129 804 AGCUGAAGAUCUGGAUGGG 1101 CCCAUCCAGAUCUUCAGCU Rh, D[1095-1113] ORF 130 805 ACCAGGCCAUGGCCAAGGA 1102 UCCUUGGCCAUGGCCUGGU Rh,D [393-411] ORF 131 806 CAUGUUCUUCAAGCCACAC 1103 GUGUGGCUUGAAGAACAUG Rh,Rb, D [838-856] ORF 132 807 CAAGAUCAACUUCCGCGAC 1104 GUCGCGGAAGUUGAUCUUGD [706-724] ORF 133 808 UCCAGCCUCAUCAUCCUCA 1105 UGAGGAUGAUGAGGCUGGA Rh,D, Rt, M [1022-1040] ORF 134 809 GCCCGAGGUCACCAAGGAC 1106GUCCUUGGUGACCUCGGGC Rh, D [784-802] ORF 135 810 UCAAGCCACACUGGGAUGA 1107UCAUCCCAGUGUGGCUUGA Rh, Rb [846-864] ORF 136 811 AGUCCAUCAACGAGUGGGC1108 GCCCACUCGUUGAUGGACU Rh, Rt, M [741-759] ORF 137 812GACUUCGUGCGCAGCAGCA 1109 UGCUGCUGCGCACGAAGUC Rh, D, M [662-680] ORF 138813 CUCUCCAGCCUCAUCAUCC 1110 GGAUGAUGAGGCUGGAGAG Rh, D, Rt, M, P[1019-1037] ORF 139 814 GCAGACCACCGACGGCAAG 1111 CUUGCCGUCGGUGGUCUGC D,Rt [763-781] ORF 140 815 AUGCAGAAGAAGGCUGUUG 1112 CAACAGCCUUCUUCUGCAU Rt[1118-1136] ORF 141 816 CAACCGUGGCUUCAUGGUG 1113 CACCAUGAAGCCACGGUUG Rh,Rt, M [889-907] ORF 142 817 UACUACGACGACGAGAAGG 1114 CCUUCUCGUCGUCGUAGUARb [962-980] ORF 143 818 GAAGGCUGUUGCCAUCUCC 1115 GGAGAUGGCAACAGCCUUC Rt[1126-1144] ORF 144 819 UCACCAAGGACGUGGAGCG 1116 CGCUCCACGUCCUUGGUGA Rh,D [792-810] ORF 145 820 CAGCUGAAGAUCUGGAUGG 1117 CCAUCCAGAUCUUCAGCUG Rh,D [1094-1112] ORF 146 821 UGGGCCUGACUGAGGCCAU 1118 AUGGCCUCAGUCAGGCCCARt [1200-1218] ORF 147 822 ACCGUGGCUUCAUGGUGAC 1119 GUCACCAUGAAGCCACGGURh, Rt, M [891-909] ORF 148 823 CAGUCCAUCAACGAGUGGG 1120CCCACUCGUUGAUGGACUG Rh, Rt, M [740-758] ORF 149 824 CCGACGGCAAGCUGCCCGA1121 UCGGGCAGCUUGCCGUCGG D [771-789] ORF 150 825 ACAAGCGCAGCGCGCUGCA1122 UGCAGCGCGCUGCGCUUGU Rh, Rt [723-741] ORF 151 826GAAACACCUGGCUGGGCUG 1123 CAGCCCAGCCAGGUGUUUC D [1183-1201] ORF 152 827AGGCUCCUGAGACACAUGG 1124 CCAUGUGUCUCAGGAGCCU D [1525-1543] 3′UTR 153 828CAAGGACGUGGAGCGCACG 1125 CGUGCGCUCCACGUCCUUG Rh, D [796-814] ORF 154 829GCAGUCCAUCAACGAGUGG 1126 CCACUCGUUGAUGGACUGC Rh, Rt, M [739-757] ORF 155830 AGAUGGUGGACAACCGUGG 1127 CCACGGUUGUCCACCAUCU Rh, M [879-897] ORF 156831 AAGCGCAGCGCGCUGCAGU 1128 ACUGCAGCGCGCUGCGCUU Rh, Rt [725-743] ORF157 832 CAUGUCAGGCAAGAAGGAC 1129 GUCCUUCUUGCCUGACAUG Rh, D [1246-1264]ORF 158 833 CAAGCCACACUGGGAUGAG 1130 CUCAUCCCAGUGUGGCUUG Rh, Rb[847-865] ORF 159 834 AAGAUGCAGAAGAAGGCUG 1131 CAGCCUUCUUCUGCAUCUU Rh,Rt, M [1115-1133] ORF 160 835 GGCCAUGGCCAAGGACCAG 1132CUGGUCCUUGGCCAUGGCC Rh, D [397-415] ORF 161 836 GUGCGCAGCAGCAAGCAGC 1133GCUGCUUGCUGCUGCGCAC Rh, D [668-686] ORF 162 837 CAACUGCGAGCACUCCAAG 1134CUUGGAGUGCUCGCAGUUG Rh, D [691-709] ORF 163 838 UACAACUGCGAGCACUCCA 1135UGGAGUGCUCGCAGUUGUA Rh, D [689-707] ORF 164 839 CAUUGACAAGAACAAGGCC 1136GGCCUUGUUCUUGUCAAUG Rh, D [1216-1234] ORF 165 840 CAAGCAGCACUACAACUGC1137 GCAGUUGUAGUGCUGCUUG Rh, D [679-697] ORF 166 841 GUGUUCCACGCCACCGCCU1138 AGGCGGUGGCGUGGAACAC D [1280-1298] ORF 167 842 CCUGCUAUUCAUUGGGCGC1139 GCGCCCAAUGAAUAGCAGG D [1426-1444] ORF 168 843 GCCCACAAGCUCUCCAGCC1140 GGCUGGAGAGCUUGUGGGC Rh, D, P [1010-1028] ORF 169 844CAGCAGCAAGCAGCACUAC 1141 GUAGUGCUGCUUGCUGCUG Rh, D [673-691] ORF 170 845UGAUGCACCGGACAGGCCU 1142 AGGCCUGUCCGGUGCAUCA Rh, Rb, Rt, M, P [936-954]ORF 171 846 UCAACUUCCGCGACAAGCG 1143 CGCUUGUCGCGGAAGUUGA D [711-729] ORF172 847 UCAGGCAAGAAGGACCUGU 1144 ACAGGUCCUUCUUGCCUGA Rh, D [1250-1268]ORF 173 848 ACUUCGUGCGCAGCAGCAA 1145 UUGCUGCUGCGCACGAAGU Rh, D, M[663-681] ORF 174 849 ACAACCGUGGCUUCAUGGU 1146 ACCAUGAAGCCACGGUUGU Rh,Rt, M [888-906] ORF 175 850 AAGGCUGUUGCCAUCUCCU 1147 AGGAGAUGGCAACAGCCUUD, Rt [1127-1145] ORF 176 851 GCAGCUGCGCGACGAGGAG 1148CUCCUCGUCGCGCAGCUGC Rh, D [529-547] ORF 177 852 UAUUCAUUGGGCGCCUGGU 1149ACCAGGCGCCCAAUGAAUA D [1431-1449] ORF 178 853 UCCACCACAAGAUGGUGGA 1150UCCACCAUCUUGUGGUGGA Rh, Rb, D, P [870-888] ORF 179 854CCCUGGCCCACAAGCUCUC 1151 GAGAGCUUGUGGGCCAGGG Rh, D, P [1005-1023] ORF180 855 ACCAGGACAUCUACGGGCG 1152 CGCCCGUAGAUGUCCUGGU D, Rt [1329-1347]ORF 181 856 GAUGAUGCACCGGACAGGC 1153 GCCUGUCCGGUGCAUCAUC Rh, Rb, Rt, M[934-952] ORF 182 857 CAACGCCAUGUUCUUCAAG 1154 CUUGAAGAACAUGGCGUUG Rh,Rb, P [832-850] ORF 183 858 ACGGCAAGCUGCCCGAGGU 1155 ACCUCGGGCAGCUUGCCGURh, D [774-792] ORF 184 859 CAGCGCGCUGCAGUCCAUC 1156 GAUGGACUGCAGCGCGCUGRh, Rt [730-748] ORF 185 860 CCCAAGGGUGUGGUGGAGG 1157CCUCCACCACACCCUUGGG Rh, D [1148-1166] ORF 186 861 CAUGGCCAAGGACCAGGCA1158 UGCCUGGUCCUUGGCCAUG Rh, D [400-418] ORF 187 862 CUCCAGCCUCAUCAUCCUC1159 GAGGAUGAUGAGGCUGGAG Rh, D, Rt, M [1021-1039] ORF 188 863UCUACGGGCGCGAGGAGCU 1160 AGCUCCUCGCGCCCGUAGA D, M [1338-1356] ORF 189864 GGCCCACAAGCUCUCCAGC 1161 GCUGGAGAGCUUGUGGGCC Rh, D, P [1009-1027]ORF 190 865 GUCAGGCAAGAAGGACCUG 1162 CAGGUCCUUCUUGCCUGAC Rh, D[1249-1267] ORF 191 866 CAUCUACGGGCGCGAGGAG 1163 CUCCUCGCGCCCGUAGAUG D,M [1336-1354] ORF 192 867 CGUGCGCAGCAGCAAGCAG 1164 CUGCUUGCUGCUGCGCACGRh, D, M [667-685] ORF 193 868 AGCCUCAUCAUCCUCAUGC 1165GCAUGAGGAUGAUGAGGCU Rh, D, Rt, M [1025-1043] ORF 194 869UUCAAGCCACACUGGGAUG 1166 CAUCCCAGUGUGGCUUGAA Rh, Rb [845-863] ORF 195870 AAGAAGGCUGUUGCCAUCU 1167 AGAUGGCAACAGCCUUCUU Rt [1124-1142] ORF 196871 GGUGUGGUGGAGGUGACCC 1168 GGGUCACCUCCACCACACC Rh, D [1154-1172] ORF197 872 GAGGUGACCCAUGACCUGC 1169 GCAGGUCAUGGGUCACCUC Rh, Rt, M[1163-1181] ORF 198 873 GUGGAGGUGACCCAUGACC 1170 GGUCAUGGGUCACCUCCAC Rh,Rt, M [1160-1178] ORF 199 874 CACAAGAUGGUGGACAACC 1171GGUUGUCCACCAUCUUGUG Rh, Rb, M, P [875-893] ORF 200 875CUGGCCCACAAGCUCUCCA 1172 UGGAGAGCUUGUGGGCCAG Rh, D, P [1007-1025] ORF201 876 GAUGACUUCGUGCGCAGCA 1173 UGCUGCGCACGAAGUCAUC Rh, Rt, M [659-677]ORF 202 877 ACUUCCGCGACAAGCGCAG 1174 CUGCGCUUGUCGCGGAAGU D [714-732] ORF203 878 AACGCCAUGUUCUUCAAGC 1175 GCUUGAAGAACAUGGCGUU Rh, Rb, P [833-851]ORF 204 879 GGACCUGUACCUGGCCAGC 1176 GCUGGCCAGGUACAGGUCC Rh, D[1261-1279] ORF 205 880 GCGACGAGGAGGUGCACGC 1177 GCGUGCACCUCCUCGUCGC D[537-555] ORF 206 881 GCAAGCUGCCCGAGGUCAC 1178 GUGACCUCGGGCAGCUUGC Rh, D[777-795] ORF 207 882 AUUCAUUGGGCGCCUGGUC 1179 GACCAGGCGCCCAAUGAAU D[1432-1450] ORF 208 883 GAGGUCACCAAGGACGUGG 1180 CCACGUCCUUGGUGACCUC Rh,D [788-806] ORF 209 884 AAGAAGGACCUGUACCUGG 1181 CCAGGUACAGGUCCUUCUU Rh,D [1256-1274] ORF 210 885 GACAACCGUGGCUUCAUGG 1182 CCAUGAAGCCACGGUUGUCRh, Rt, M [887-905] ORF 211 886 CUGGGCCUGACUGAGGCCA 1183UGGCCUCAGUCAGGCCCAG Rt [1199-1217] ORF 212 887 CUCCAAGAUCAACUUCCGC 1184GCGGAAGUUGAUCUUGGAG Rh, D, Rt, M [703-721] ORF 213 888CAACUUCCGCGACAAGCGC 1185 GCGCUUGUCGCGGAAGUUG D [712-730] ORF 214 889CUCCCUGCUAUUCAUUGGG 1186 CCCAAUGAAUAGCAGGGAG D [1423-1441] ORF 215 890AAGCAGCACUACAACUGCG 1187 CGCAGUUGUAGUGCUGCUU Rh, D [680-698] ORF 216 891GCGCAGCAGCAAGCAGCAC 1188 GUGCUGCUUGCUGCUGCGC Rh, D [670-688] ORF 217 892CAGGCCAUGGCCAAGGACC 1189 GGUCCUUGGCCAUGGCCUG Rh, D [395-413] ORF 218 893GUACCAGGCCAUGGCCAAG 1190 CUUGGCCAUGGCCUGGUAC Rh, D [391-409] ORF 219 894CUUCGUGCGCAGCAGCAAG 1191 CUUGCUGCUGCGCACGAAG Rh, D, M [664-682] ORF 220895 CAGCACUACAACUGCGAGC 1192 GCUCGCAGUUGUAGUGCUG Rh, D [683-701] ORF 221896 UACAACUACUACGACGACG 1193 CGUCGUCGUAGUAGUUGUA Rb [956-974] ORF 222897 GAUGGUGGACAACCGUGGC 1194 GCCACGGUUGUCCACCAUC Rh, M [880-898] ORF 223898 CUACAACUGCGAGCACUCC 1195 GGAGUGCUCGCAGUUGUAG Rh, D [688-706] ORF 224899 AAGGACCUGUACCUGGCCA 1196 UGGCCAGGUACAGGUCCUU Rh, D [1259-1277] ORF225 900 GCUGCCCGAGGUCACCAAG 1197 CUUGGUGACCUCGGGCAGC Rh, D [781-799] ORF226 901 GACAUCUACGGGCGCGAGG 1198 CCUCGCGCCCGUAGAUGUC D, M [1334-1352]ORF 227 902 CCACCACAAGAUGGUGGAC 1199 GUCCACCAUCUUGUGGUGG Rh, Rb, D, P[871-889] ORF 228 903 GCGCGACGAGGAGGUGCAC 1200 GUGCACCUCCUCGUCGCGC Rh, D[535-553] ORF 229 904 CUAUUCAUUGGGCGCCUGG 1201 CCAGGCGCCCAAUGAAUAG D[1430-1448] ORF 230 905 CCAGGACAUCUACGGGCGC 1202 GCGCCCGUAGAUGUCCUGG D,Rt [1330-1348] ORF 231 906 AAGAUGGUGGACAACCGUG 1203 CACGGUUGUCCACCAUCUURh, M [878-896] ORF 232 907 CAGGACAUCUACGGGCGCG 1204 CGCGCCCGUAGAUGUCCUGD [1331-1349] ORF 233 908 UCCAAGAUCAACUUCCGCG 1205 CGCGGAAGUUGAUCUUGGA D[704-722] ORF 234 909 GUCACCAAGGACGUGGAGC 1206 GCUCCACGUCCUUGGUGAC Rh, D[791-809] ORF 235 910 CUGCCCGAGGUCACCAAGG 1207 CCUUGGUGACCUCGGGCAG Rh, D[782-800] ORF 236 911 GACCAGGACAUCUACGGGC 1208 GCCCGUAGAUGUCCUGGUC D, Rt[1328-1346] ORF 237 912 CCAUGGCCAAGGACCAGGC 1209 GCCUGGUCCUUGGCCAUGG Rh,D [399-417] ORF 238 913 CACCAAGGACGUGGAGCGC 1210 GCGCUCCACGUCCUUGGUG Rh,D [793-811] ORF 239 914 GACAAGCGCAGCGCGCUGC 1211 GCAGCGCGCUGCGCUUGUC Rh,Rt [722-740] ORF 240 915 CAAGCGCAGCGCGCUGCAG 1212 CUGCAGCGCGCUGCGCUUGRh, Rt [724-742] ORF 241 916 CAGACCACCGACGGCAAGC 1213GCUUGCCGUCGGUGGUCUG D, Rt [764-782] ORF 242 917 GACCACCGACGGCAAGCUG 1214CAGCUUGCCGUCGGUGGUC D, Rt [766-784] ORF 243 918 AGGACCUGUACCUGGCCAG 1215CUGGCCAGGUACAGGUCCU Rh, D [1260-1278] ORF 244 919 CUGCUAUUCAUUGGGCGCC1216 GGCGCCCAAUGAAUAGCAG D [1427-1445] ORF 245 920 UCAUUGGGCGCCUGGUCCG1217 CGGACCAGGCGCCCAAUGA Rh, D [1434-1452] ORF 246 921GCUGCGCGACGAGGAGGUG 1218 CACCUCCUCGUCGCGCAGC Rh, D [532-550] ORF 247 922CGGCAAGCUGCCCGAGGUC 1219 GACCUCGGGCAGCUUGCCG Rh, D [775-793] ORF 248 923CCUCAUCAUCCUCAUGCCC 1220 GGGCAUGAGGAUGAUGAGG Rh, D, Rt, M [1027-1045]ORF 249 924 CCAGGCCAUGGCCAAGGAC 1221 GUCCUUGGCCAUGGCCUGG Rh, D [394-412]ORF 250 925 GCCAUGGCCAAGGACCAGG 1222 CCUGGUCCUUGGCCAUGGC Rh, D [398-416]ORF 251 926 CCACCGACGGCAAGCUGCC 1223 GGCAGCUUGCCGUCGGUGG D, Rt [768-786]ORF 252 927 AUGGUGGACAACCGUGGCU 1224 AGCCACGGUUGUCCACCAU Rh, M [881-899]ORF 253 928 CUUCCGCGACAAGCGCAGC 1225 GCUGCGCUUGUCGCGGAAG D [715-733] ORF254 929 CGCGACGAGGAGGUGCACG 1226 CGUGCACCUCCUCGUCGCG Rh, D [536-554] ORF255 930 UGGCCCACAAGCUCUCCAG 1227 CUGGAGAGCUUGUGGGCCA Rh, D, P[1008-1026] ORF 256 931 GAGCAGCUGCGCGACGAGG 1228 CCUCGUCGCGCAGCUGCUC Rh,D [527-545] ORF 257 932 UGACCAGGACAUCUACGGG 1229 CCCGUAGAUGUCCUGGUCA Rt[1327-1345] ORF 258 933 ACCACCGACGGCAAGCUGC 1230 GCAGCUUGCCGUCGGUGGU D,Rt [767-785] ORF 259 934 GAAGGACCUGUACCUGGCC 1231 GGCCAGGUACAGGUCCUUCRh, D [1258-1276] ORF 260 935 CAUUGGGCGCCUGGUCCGG 1232CCGGACCAGGCGCCCAAUG Rh, D [1435-1453] ORF 261 936 AUGCACCGGACAGGCCUCU1233 AGAGGCCUGUCCGGUGCAU Rh, Rb, Rt, P [938-956] ORF 262 937AUCAACUUCCGCGACAAGC 1234 GCUUGUCGCGGAAGUUGAU D [710-728] ORF 263 938CAGCUGCGCGACGAGGAGG 1235 CCUCCUCGUCGCGCAGCUG Rh, D [530-548] ORF 264 939CAGAAACACCUGGCUGGGC 1236 GCCCAGCCAGGUGUUUCUG D [1181-1199] ORF 265 940CUACGGGCGCGAGGAGCUG 1237 CAGCUCCUCGCGCCCGUAG D, M [1339-1357] ORF 266941 CGACGAGGAGGUGCACGCC 1238 GGCGUGCACCUCCUCGUCG D [538-556] ORF 267 942UUUGACCAGGACAUCUACG 1239 CGUAGAUGUCCUGGUCAAA Rt [1325-1343] ORF 268 943GUCCAUCAACGAGUGGGCC 1240 GGCCCACUCGUUGAUGGAC Rh, Rt, M [742-760] ORF 269944 AUGACUUCGUGCGCAGCAG 1241 CUGCUGCGCACGAAGUCAU Rh, Rt, M [660-678] ORF270 945 UCCCUGCUAUUCAUUGGGC 1242 GCCCAAUGAAUAGCAGGGA D [1424-1442] ORF271 946 CUGCGCGACGAGGAGGUGC 1243 GCACCUCCUCGUCGCGCAG Rh, D [533-551] ORF272 947 CAAGCUGCCCGAGGUCACC 1244 GGUGACCUCGGGCAGCUUG Rh, D [778-796] ORF273 948 AAGCUGCCCGAGGUCACCA 1245 UGGUGACCUCGGGCAGCUU Rh, D [779-797] ORF274 949 UUCUUCAAGCCACACUGGG 1246 CCCAGUGUGGCUUGAAGAA Rh, Rb, D [842-860]ORF 275 950 ACACCUGGCUGGGCUGGGC 1247 GCCCAGCCCAGCCAGGUGU D [1186-1204]ORF 276 951 UCCAUCAACGAGUGGGCCG 1248 CGGCCCACUCGUUGAUGGA Rt, M [743-761]ORF 277 952 AUCUACGGGCGCGAGGAGC 1249 GCUCCUCGCGCCCGUAGAU D, M[1337-1355] ORF 278 953 UCGUGCGCAGCAGCAAGCA 1250 UGCUUGCUGCUGCGCACGA Rh,D, M [666-684] ORF 279 954 CGACGGCAAGCUGCCCGAG 1251 CUCGGGCAGCUUGCCGUCGD [772-790] ORF 280 955 UUCAUUGGGCGCCUGGUCC 1252 GGACCAGGCGCCCAAUGAA Rh,D [1433-1451] ORF 281 956 UUGACCAGGACAUCUACGG 1253 CCGUAGAUGUCCUGGUCAARt [1326-1344] ORF 282 957 CCUGGCCCACAAGCUCUCC 1254 GGAGAGCUUGUGGGCCAGGRh, D, P [1006-1024] ORF 283 958 UGACUUCGUGCGCAGCAGC 1255GCUGCUGCGCACGAAGUCA Rh, Rt, M [661-679] ORF 284 959 AUGAUGCACCGGACAGGCC1256 GGCCUGUCCGGUGCAUCAU Rh, Rb, Rt, M, P [935-953] ORF 285 960CACCGACGGCAAGCUGCCC 1257 GGGCAGCUUGCCGUCGGUG D, Rt [769-787] ORF 286 961GACGGCAAGCUGCCCGAGG 1258 CCUCGGGCAGCUUGCCGUC Rh, D [773-791] ORF 287 962UACCAGGCCAUGGCCAAGG 1259 CCUUGGCCAUGGCCUGGUA Rh, D [392-410] ORF 288 963UCCGCGACAAGCGCAGCGC 1260 GCGCUGCGCUUGUCGCGGA D [717-735] ORF 289 964UUCCGCGACAAGCGCAGCG 1261 CGCUGCGCUUGUCGCGGAA D [716-734] ORF 290 965AAGGACGUGGAGCGCACGG 1262 CCGUGCGCUCCACGUCCUU Rh, D [797-815] ORF 291 966UUCCACCACAAGAUGGUGG 1263 CCACCAUCUUGUGGUGGAA Rh, Rb, D, P [869-887] ORF292 967 UACGGGCGCGAGGAGCUGC 1264 GCAGCUCCUCGCGCCCGUA D, M [1340-1358]ORF 293 968 AAACACCUGGCUGGGCUGG 1265 CCAGCCCAGCCAGGUGUUU D [1184-1202]ORF 294 969 AACACCUGGCUGGGCUGGG 1266 CCCAGCCCAGCCAGGUGUU D [1185-1203]ORF 295 970 AUUGGGCGCCUGGUCCGGC 1267 GCCGGACCAGGCGCCCAAU Rh, D[1436-1454] ORF 296 971 ACCGACGGCAAGCUGCCCG 1268 CGGGCAGCUUGCCGUCGGU D[770-788] ORF 297 972 UUCGUGCGCAGCAGCAAGC 1269 GCUUGCUGCUGCGCACGAA Rh,D, M [665-683] ORF

TABLE D SERPINH1 Active 18 + 1-mer siRNAs human- 32454740 No. SEQ IDSense siRNA SEQ ID AntiSense siRNA Other Sp ORF: 230-1486 1 1270AGCCUUUGUUGCUAUCAAA 1849 UUUGAUAGCAACAAAGGCU Rh [2117-2135] 3′UTR 2 1271GCCUAAGGGUGACAAGAUA 1850 UAUCUUGUCACCCUUAGGC Rh [1453-1471] ORF 3 1272GGCCUAAGGGUGACAAGAA 1851 UUCUUGUCACCCUUAGGCC Rh [1452-1470] ORF 4 1273CCUCAAUCAGUAUUCAUAA 1852 UUAUGAAUACUGAUUGAGG [1774-1792] 3′UTR 5 1274GGCGGAUUGAGAAGGAGCA 1853 UGCUCCUUCUCAAUCCGCC [1973-1991] 3′UTR 6 1275GGCAGUGGAGAACAUCCUA 1854 UAGGAUGUUCUCCACUGCC Rh [415-433] ORF 7 1276GGGUCAGCCAGCCCUCUUA 1855 UAAGAGGGCUGGCUGACCC Rh [1839-1857] 3′UTR 8 1277GGGUGACAAGAUGCGAGAA 1856 UUCUCGCAUCUUGUCACCC Rh, D [1459-1477] ORF 91278 GGACCAGGCAGUGGAGAAA 1857 UUUCUCCACUGCCUGGUCC Rh [409-427] ORF 101279 GAGACACAUGGGUGCUAUA 1858 UAUAGCACCCAUGUGUCUC Rh, D, Rt, M[1533-1551] 3′UTR 11 1280 GUUGGAGCGUGGAAAAAAA 1859 UUUUUUUCCACGCUCCAAC[2191-2208] 3′UTR 12 1281 GGAACAUGAGCCUUUGUUA 1860 UAACAAAGGCUCAUGUUCCRh [2109-2127] 3′UTR 13 1282 GCCAUGUUCUUCAAGCCAA 1861UUGGCUUGAAGAACAUGGC Rh, Rb, D [836-854] ORF 14 1283 GGAUUGAGAAGGAGCUCCA1862 UGGAGCUCCUUCUCAAUCC [1976-1994] 3′UTR 15 1284 GGGAUGAACUUUUUGUUUA1863 UAAACAAAAAGUUCAUCCC Rh [2048-2066] 3′UTR 16 1285GCCGCAGUGAGGCGGAUUA 1864 UAAUCCGCCUCACUGCGGC [1963-1981] 3′UTR 17 1286GGACCUUCCCAGCUAGAAA 1865 UUUCUAGCUGGGAAGGUCC Rh [1639-1657] 3′UTR 181287 GACCUUCCCAGCUAGAAUA 1866 UAUUCUAGCUGGGAAGGUC Rh [1640-1658] 3′UTR19 1288 CCUGUGAGACCAAAUUGAA 1867 UUCAAUUUGGUCUCACAGG Rh [1814-1832]3′UTR 20 1289 UGGAGAACAUCCUGGUGUA 1868 UACACCAGGAUGUUCUCCA Rh [420-438]ORF 21 1290 GCCUUUGUUGCUAUCAAUA 1869 UAUUGAUAGCAACAAAGGC Rh [2118-2136]3′UTR 22 1291 CCGCCUUUGAGUUGGACAA 1870 UUGUCCAACUCAAAGGCGG Rh[1293-1311] ORF 23 1292 CAGGCAGUGGAGAACAUCA 1871 UGAUGUUCUCCACUGCCUG Rh[413-431] ORF 24 1293 CACCUGUGAGACCAAAUUA 1872 UAAUUUGGUCUCACAGGUG Rh[1812-1830] 3′UTR 25 1294 GGGAAGAUGCAGAAGAAGA 1873 UCUUCUUCUGCAUCUUCCCRh, Rb, Rt [1112-1130] ORF 26 1295 GGCCAUUGACAAGAACAAA 1874UUUGUUCUUGUCAAUGGCC Rh, D [1213-1231] ORF 27 1296 GCCUUUGAGUUGGACACAA1875 UUGUGUCCAACUCAAAGGC Rh [1295-1313] ORF 28 1297 AGCGGACCUUCCCAGCUAA1876 UUAGCUGGGAAGGUCCGCU Rh [1636-1654] 3′UTR 29 1298GAAGAAGGCUGUUGCCAUA 1877 UAUGGCAACAGCCUUCUUC Rt [1123-1141] ORF 30 1299ACAAGAUGCGAGACGAGUA 1878 UACUCGUCUCGCAUCUUGU Rh, Rt [1464-1482] ORF 311300 GAGGCGGAUUGAGAAGGAA 1879 UUCCUUCUCAAUCCGCCUC [1971-1989] 3′UTR 321301 GGACAACCGUGGCUUCAUA 1880 UAUGAAGCCACGGUUGUCC Rh, M [886-904] ORF 331302 CAUAUUUAUAGCCAGGUAA 1881 UUACCUGGCUAUAAAUAUG Rh [1788-1806] 3′UTR34 1303 CGACGACGAGAAGGAAAAA 1882 UUUUUCCUUCUCGUCGUCG [967-985] ORF 351304 CUCACCUGUGAGACCAAAA 1883 UUUUGGUCUCACAGGUGAG Rh [1810-1828] 3′UTR36 1305 GCGGCUCCCUGCUAUUCAA 1884 UUGAAUAGCAGGGAGCCGC [1419-1437] ORF 371306 AGAACAUCCUGGUGUCACA 1885 UGUGACACCAGGAUGUUCU [423-441] ORF 38 1307CACACUGGGAUGAGAAAUA 1886 UAUUUCUCAUCCCAGUGUG Rh [852-870] ORF 39 1308GCUAGAAUUCACUCCACUA 1887 UAGUGGAGUGAAUUCUAGC Rh [1650-1668] 3′UTR 401309 CCUUCAUCUUCCUAGUGCA 1888 UGCACUAGGAAGAUGAAGG [1389-1407] ORF 411310 UGCUAUCAAUCCAAGAACA 1889 UGUUCUUGGAUUGAUAGCA Rh [2126-2144] 3′UTR42 1311 GGAAGAUGCAGAAGAAGGA 1890 UCCUUCUUCUGCAUCUUCC Rh, Rb, Rt[1113-1131] ORF 43 1312 CAUGAGCCUUUGUUGCUAA 1891 UUAGCAACAAAGGCUCAUG Rh[2113-2131] 3′UTR 44 1313 GCGGAUUGAGAAGGAGCUA 1892 UAGCUCCUUCUCAAUCCGC[1974-1992] 3′UTR 45 1314 UGCAGUCCAUCAACGAGUA 1893 UACUCGUUGAUGGACUGCARh, Rt, M [738-756] ORF 46 1315 GCACUGCGGAGAAGUUGAA 1894UUCAACUUCUCCGCAGUGC [321-339] ORF 47 1316 CCAGGCAGUGGAGAACAUA 1895UAUGUUCUCCACUGCCUGG Rh [412-430] ORF 48 1317 GGCAAGAAGGACCUGUACA 1896UGUACAGGUCCUUCUUGCC Rh, D, M [1253-1271] ORF 49 1318 CUCUACAACUACUACGACA1897 UGUCGUAGUAGUUGUAGAG Rb [953-971] ORF 50 1319 CUUCCCAGCUAGAAUUCAA1898 UUGAAUUCUAGCUGGGAAG Rh [1643-1661] 3′UTR 51 1320AGGCGGAUUGAGAAGGAGA 1899 UCUCCUUCUCAAUCCGCCU [1972-1990] 3′UTR 52 1321GGUCCUAUACCGUGGGUGA 1900 UCACCCACGGUAUAGGACC Rh [912-930] ORF 53 1322GCAAGAAGGACCUGUACCA 1901 UGGUACAGGUCCUUCUUGC Rh, D, M [1254-1272] ORF 541323 CCGUGGGUGUCAUGAUGAA 1902 UUCAUCAUGACACCCACGG Rh [921-939] ORF 551324 GAUGCGAGACGAGUUAUAA 1903 UUAUAACUCGUCUCGCAUC Rh [1468-1486] ORF 561325 GGCAGUGCUGAGCGCCGAA 1904 UUCGGCGCUCAGCACUGCC [511-529] ORF 57 1326CAGCUAGAAUUCACUCCAA 1905 UUGGAGUGAAUUCUAGCUG Rh [1648-1666] 3′UTR 581327 GAGCUUCGCUGAUGACUUA 1906 UAAGUCAUCAGCGAAGCUC Rh [649-667] ORF 591328 CUUUGAGUUGGACACAGAA 1907 UUCUGUGUCCAACUCAAAG Rh [1297-1315] ORF 601329 GGUGGACAACCGUGGCUUA 1908 UAAGCCACGGUUGUCCACC Rh, M [883-901] ORF 611330 GCCUCAUCAUCCUCAUGCA 1909 UGCAUGAGGAUGAUGAGGC Rh, D, Rt, M[1026-1044] ORF 62 1331 ACCAGGCAGUGGAGAACAA 1910 UUGUUCUCCACUGCCUGGU Rh[411-429] ORF 63 1332 CCUGCCUCAAUCAGUAUUA 1911 UAAUACUGAUUGAGGCAGG[1770-1788] 3′UTR 64 1333 GAUCAAGCCUGCCUCAAUA 1912 UAUUGAGGCAGGCUUGAUCRh [1763-1781] 3′UTR 65 1334 CAGACUCUGGUCAAGAAGA 1913UCUUCUUGACCAGAGUCUG Rh [2011-2029] 3′UTR 66 1335 CGCGCUGCAGUCCAUCAAA1914 UUUGAUGGACUGCAGCGCG Rh, Rt [733-751] ORF 67 1336CUGGCACUGCGGAGAAGUA 1915 UACUUCUCCGCAGUGCCAG [318-336] ORF 68 1337CCAGCUCUAUCCCAACCUA 1916 UAGGUUGGGAUAGAGCUGG [1885-1903] 3′UTR 69 1338AGGGUGUGGUGGAGGUGAA 1917 UUCACCUCCACCACACCCU Rh, D [1152-1170] ORF 701339 AGUGAGGCGGAUUGAGAAA 1918 UUUCUCAAUCCGCCUCACU [1968-1986] 3′UTR 711340 CGGACAGGCCUCUACAACA 1919 UGUUGUAGAGGCCUGUCCG Rh, Rb, Rt, P[944-962] ORF 72 1341 CGACGAGAAGGAAAAGCUA 1920 UAGCUUUUCCUUCUCGUCG Rh[970-988] ORF 73 1342 AGGCCAAGGCAGUGCUGAA 1921 UUCAGCACUGCCUUGGCCU Rh[504-522] ORF 74 1343 GCCUCAGGGUGCACACAGA 1922 UCUGUGUGCACCCUGAGGC[1488-1506] 3′UTR 75 1344 GGAUGAGAAAUUCCACCAA 1923 UUGGUGGAAUUUCUCAUCCRh [859-877] ORF 76 1345 AGAAGGAAAAGCUGCAAAA 1924 UUUUGCAGCUUUUCCUUCU Rh[975-993] ORF 77 1346 AGCUCUAUCCCAACCUCUA 1925 UAGAGGUUGGGAUAGAGCU Rh[1887-1905] 3′UTR 78 1347 UGACAAGAUGCGAGACGAA 1926 UUCGUCUCGCAUCUUGUCARh [1462-1480] ORF 79 1348 AGAAGGAGCUCCCAGGAGA 1927 UCUCCUGGGAGCUCCUUCU[1982-2000] 3′UTR 80 1349 CCUUCUCACCUGUGAGACA 1928 UGUCUCACAGGUGAGAAGGRh [1806-1824] 3′UTR 81 1350 GGCUUCUGGGCAGACUCUA 1929UAGAGUCUGCCCAGAAGCC Rh [2001-2019] 3′UTR 82 1351 CCAGCCUCAUCAUCCUCAA1930 UUGAGGAUGAUGAGGCUGG Rh, D, Rt, M [1023-1041] ORF 83 1352CCAAAGGCUCCUGAGACAA 1931 UUGUCUCAGGAGCCUUUGG [1521-1539] 3′UTR 84 1353GGACCUGGGCCAUAGUCAA 1932 UUGACUAUGGCCCAGGUCC [1722-1740] 3′UTR 85 1354GGGUGUCAUGAUGAUGCAA 1933 UUGCAUCAUCAUGACACCC Rh [925-943] ORF 86 1355GUACCAGCCUUGGAUACUA 1934 UAGUAUCCAAGGCUGGUAC Rh [1572-1590] 3′UTR 871356 GGCUGUUGCCAUCUCCUUA 1935 UAAGGAGAUGGCAACAGCC [1129-1147] ORF 881357 CGCAGUGAGGCGGAUUGAA 1936 UUCAAUCCGCCUCACUGCG [1965-1983] 3′UTR 891358 CCAAGGACGUGGAGCGCAA 1937 UUGCGCUCCACGUCCUUGG Rh, D [795-813] ORF 901359 GGCUCCUGAGACACAUGGA 1938 UCCAUGUGUCUCAGGAGCC D [1526-1544] 3′UTR 911360 GCUGCAGUCCAUCAACGAA 1939 UUCGUUGAUGGACUGCAGC Rh, Rt [736-754] ORF92 1361 CCAGGUACCUUCUCACCUA 1940 UAGGUGAGAAGGUACCUGG Rh [1799-1817]3′UTR 93 1362 GCAGCGCGCUGCAGUCCAA 1941 UUGGACUGCAGCGCGCUGC Rh, Rt[729-747] ORF 94 1363 GAGACCAAAUUGAGCUAGA 1942 UCUAGCUCAAUUUGGUCUC Rh[1819-1837] 3′UTR 95 1364 GCCGCCGAGGUGAAGAAAA 1943 UUUUCUUCACCUCGGCGGC[281-299] ORF 96 1365 GCAGACUCUGGUCAAGAAA 1944 UUUCUUGACCAGAGUCUGC Rh[2010-2028] 3′UTR 97 1366 CUAGAAUUCACUCCACUUA 1945 UAAGUGGAGUGAAUUCUAGRh [1651-1669] 3′UTR 98 1367 GCAGUGGAGAACAUCCUGA 1946UCAGGAUGUUCUCCACUGC Rh [416-434] ORF 99 1368 CGCAUGUCAGGCAAGAAGA 1947UCUUCUUGCCUGACAUGCG Rh, D [1244-1262] ORF 100 1369 CGGAUUGAGAAGGAGCUCA1948 UGAGCUCCUUCUCAAUCCG [1975-1993] 3′UTR 101 1370 AGGUGAGGUACCAGCCUUA1949 UAAGGCUGGUACCUCACCU Rh [1565-1583] 3′UTR 102 1371CCACACUGGGAUGAGAAAA 1950 UUUUCUCAUCCCAGUGUGG Rh [851-869] ORF 103 1372GCCAUUGACAAGAACAAGA 1951 UCUUGUUCUUGUCAAUGGC Rh, D [1214-1232] ORF 1041373 GCGCUGCAGUCCAUCAACA 1952 UGUUGAUGGACUGCAGCGC Rh, Rt [734-752] ORF105 1374 CUCCCAACUAUAAAACUAA 1953 UUAGUUUUAUAGUUGGGAG Rh [1903-1921]3′UTR 106 1375 GGUGACAAGAUGCGAGACA 1954 UGUCUCGCAUCUUGUCACC Rh[1460-1478] ORF 107 1376 GGCCGACUUGUCACGCAUA 1955 UAUGCGUGACAAGUCGGCC Rh[1231-1249] ORF 108 1377 CCUAAGGGUGACAAGAUGA 1956 UCAUCUUGUCACCCUUAGG Rh[1454-1472] ORF 109 1378 UGAGACACAUGGGUGCUAA 1957 UUAGCACCCAUGUGUCUCARh, D, Rt, M [1532-1550] 3′UTR 110 1379 GGGUGGAAAAACAGACCGA 1958UCGGUCUGUUUUUCCACCC [1601-1619] 3′UTR 111 1380 GGUGGAGGUGACCCAUGAA 1959UUCAUGGGUCACCUCCACC Rh, Rt, M [1159-1177] ORF 112 1381CUUUGACCAGGACAUCUAA 1960 UUAGAUGUCCUGGUCAAAG Rh, Rt [1324-1342] ORF 1131382 GAACAUGAGCCUUUGUUGA 1961 UCAACAAAGGCUCAUGUUC Rh [2110-2128] 3′UTR114 1383 AGCCUUGGAUACUCCAUGA 1962 UCAUGGAGUAUCCAAGGCU Rh [1577-1595]3′UTR 115 1384 GGAGGUGACCCAUGACCUA 1963 UAGGUCAUGGGUCACCUCC Rh, Rt, M[1162-1180] ORF 116 1385 AGAUCAAGCCUGCCUCAAA 1964 UUUGAGGCAGGCUUGAUCU Rh[1762-1780] 3′UTR 117 1386 GCCCAAGGGUGUGGUGGAA 1965 UUCCACCACACCCUUGGGCRh, D [1147-1165] ORF 118 1387 AGAACAAGGCCGACUUGUA 1966UACAAGUCGGCCUUGUUCU Rh [1224-1242] ORF 119 1388 GUGGCUUCAUGGUGACUCA 1967UGAGUCACCAUGAAGCCAC Rh [894-912] ORF 120 1389 CUCCUGAGACACAUGGGUA 1968UACCCAUGUGUCUCAGGAG D [1528-1546] 3′UTR 121 1390 CAGCCUUGGAUACUCCAUA1969 UAUGGAGUAUCCAAGGCUG Rh [1576-1594] 3′UTR 122 1391AAGGCUCCUGAGACACAUA 1970 UAUGUGUCUCAGGAGCCUU D [1524-1542] 3′UTR 1231392 AGAAGAAGGCUGUUGCCAA 1971 UUGGCAACAGCCUUCUUCU Rt [1122-1140] ORF 1241393 CUACUACGACGACGAGAAA 1972 UUUCUCGUCGUCGUAGUAG Rb [961-979] ORF 1251394 CCUUUGUUGCUAUCAAUCA 1973 UGAUUGAUAGCAACAAAGG Rh [2119-2137] 3′UTR126 1395 AGGCAGUGGAGAACAUCCA 1974 UGGAUGUUCUCCACUGCCU Rh [414-432] ORF127 1396 CCAUCACGUGGAGCCUCUA 1975 UAGAGGCUCCACGUGAUGG Rh [1045-1063] ORF128 1397 AGCUCUCCAGCCUCAUCAA 1976 UUGAUGAGGCUGGAGAGCU Rh, D, Rt,[1017-1035] M, P ORF 129 1398 GGCUCCCUGCUAUUCAUUA 1977UAAUGAAUAGCAGGGAGCC D [1421-1439] ORF 130 1399 GGGAACAUGAGCCUUUGUA 1978UACAAAGGCUCAUGUUCCC Rh [2108-2126] 3′UTR 131 1400 GGGCCAUAGUCAUUCUGCA1979 UGCAGAAUGACUAUGGCCC [1728-1746] 3′UTR 132 1401 CCAAAGAGCAGCUGAAGAA1980 UUCUUCAGCUGCUCUUUGG Rh, Rb, P [1086-1104] ORF 133 1402GACGAGAAGGAAAAGCUGA 1981 UCAGCUUUUCCUUCUCGUC Rh [971-989] ORF 134 1403GGGCUUCUGGGCAGACUCA 1982 UGAGUCUGCCCAGAAGCCC Rh [2000-2018] 3′UTR 1351404 CAAGGACCAGGCAGUGGAA 1983 UUCCACUGCCUGGUCCUUG Rh [406-424] ORF 1361405 CUGUGAGACCAAAUUGAGA 1984 UCUCAAUUUGGUCUCACAG Rh [1815-1833] 3′UTR137 1406 GACUGAGGCCAUUGACAAA 1985 UUUGUCAAUGGCCUCAGUC Rh [1207-1225] ORF138 1407 GACUUGUCACGCAUGUCAA 1986 UUGACAUGCGUGACAAGUC Rh [1235-1253] ORF139 1408 GAGGUGAGGUACCAGCCUA 1987 UAGGCUGGUACCUCACCUC [1564-1582] 3′UTR140 1409 CAGAUACCAUGAUGCUGAA 1988 UUCAGCAUCAUGGUAUCUG Rh [1681-1699]3′UTR 141 1410 AGGCAAGAAGGACCUGUAA 1989 UUACAGGUCCUUCUUGCCU Rh, D[1252-1270] ORF 142 1411 CUGGGAUGAGAAAUUCCAA 1990 UUGGAAUUUCUCAUCCCAG Rh[856-874] ORF 143 1412 AGGUACCAGCCUUGGAUAA 1991 UUAUCCAAGGCUGGUACCU Rh[1570-1588] 3′UTR 144 1413 CAGCCAGCCCUCUUCUGAA 1992 UUCAGAAGAGGGCUGGCUG[1843-1861] 3′UTR 145 1414 GUGUCAUGAUGAUGCACCA 1993 UGGUGCAUCAUCAUGACACRh [927-945] ORF 146 1415 CCUCUACAACUACUACGAA 1994 UUCGUAGUAGUUGUAGAGGRb, D [952-970] ORF 147 1416 CCGCCGAGGUGAAGAAACA 1995UGUUUCUUCACCUCGGCGG Rh [282-300] ORF 148 1417 GCUAUCAAUCCAAGAACUA 1996UAGUUCUUGGAUUGAUAGC Rh [2127-2145] 3′UTR 149 1418 AGCCUGCCUCAAUCAGUAA1997 UUACUGAUUGAGGCAGGCU [1768-1786] 3′UTR 150 1419 GGUCCGGCCUAAGGGUGAA1998 UUCACCCUUAGGCCGGACC Rh [1447-1465] ORF 151 1420 GAAGGAAAAGCUGCAAAUA1999 UAUUUGCAGCUUUUCCUUC Rh [976-994] ORF 152 1421 GGCCUCUACAACUACUACA2000 UGUAGUAGUUGUAGAGGCC Rb, D [950-968] ORF 153 1422UGUUCUUCAAGCCACACUA 2001 UAGUGUGGCUUGAAGAACA Rh, Rb, D [840-858] ORF 1541423 GGCCAAGGCAGUGCUGAGA 2002 UCUCAGCACUGCCUUGGCC Rh [505-523] ORF 1551424 AGAAAUUCCACCACAAGAA 2003 UUCUUGUGGUGGAAUUUCU Rh [864-882] ORF 1561425 CUGCAGUCCAUCAACGAGA 2004 UCUCGUUGAUGGACUGCAG Rh, Rt, M [737-755]ORF 157 1426 CCAGCGUGUUCCACGCCAA 2005 UUGGCGUGGAACACGCUGG [1275-1293]ORF 158 1427 GCUCCCUCCUGCUUCUCAA 2006 UUGAGAAGCAGGAGGGAGC [234-252] ORF159 1428 CCGGACAGGCCUCUACAAA 2007 UUUGUAGAGGCCUGUCCGG Rh, Rb, Rt, P[943-961] ORF 160 1429 CCCAUCACGUGGAGCCUCA 2008 UGAGGCUCCACGUGAUGGG Rh[1044-1062] ORF 161 1430 CCGGCCUAAGGGUGACAAA 2009 UUUGUCACCCUUAGGCCGG Rh[1450-1468] ORF 162 1431 CCUAUACCGUGGGUGUCAA 2010 UUGACACCCACGGUAUAGGRh, D, P [915-933] ORF 163 1432 CAGUGGAGAACAUCCUGGA 2011UCCAGGAUGUUCUCCACUG Rh [417-435] ORF 164 1433 CACUGGGAUGAGAAAUUCA 2012UGAAUUUCUCAUCCCAGUG Rh [854-872] ORF 165 1434 AUCCAAAGGCUCCUGAGAA 2013UUCUCAGGAGCCUUUGGAU [1519-1537] 3′UTR 166 1435 UGAGAAAUUCCACCACAAA 2014UUUGUGGUGGAAUUUCUCA Rh [862-880] ORF 167 1436 GGUGGAAAAACAGACCGGA 2015UCCGGUCUGUUUUUCCACC [1602-1620] 3′UTR 168 1437 GCUGGGCAGCCGACUGUAA 2016UUACAGUCGGCUGCCCAGC [616-634] ORF 169 1438 CCAUAGUCAUUCUGCCUGA 2017UCAGGCAGAAUGACUAUGG [1731-1749] 3′UTR 170 1439 GCACCGGACAGGCCUCUAA 2018UUAGAGGCCUGUCCGGUGC Rh, Rb, Rt, P [940-958] ORF 171 1440GUUGGACACAGAUGGCAAA 2019 UUUGCCAUCUGUGUCCAAC [1303-1321] ORF 172 1441GCCUGCCUCAAUCAGUAUA 2020 UAUACUGAUUGAGGCAGGC [1769-1787] 3′UTR 173 1442GAUCAACUUCCGCGACAAA 2021 UUUGUCGCGGAAGUUGAUC D [709-727] ORF 174 1443GGCCGCAGUGAGGCGGAUA 2022 UAUCCGCCUCACUGCGGCC [1962-1980] 3′UTR 175 1444CUGCGGAGAAGUUGAGCCA 2023 UGGCUCAACUUCUCCGCAG [324-342] ORF 176 1445GCAUCCAAAGGCUCCUGAA 2024 UUCAGGAGCCUUUGGAUGC [1517-1535] 3′UTR 177 1446GCUUCUGGGCAGACUCUGA 2025 UCAGAGUCUGCCCAGAAGC Rh [2002-2020] 3′UTR 1781447 CCAGCCCUCUUCUGACACA 2026 UGUGUCAGAAGAGGGCUGG [1846-1864] 3′UTR 1791448 GCUCUAUCCCAACCUCUCA 2027 UGAGAGGUUGGGAUAGAGC Rh [1888-1906] 3′UTR180 1449 GGACGUGGAGCGCACGGAA 2028 UUCCGUGCGCUCCACGUCC Rh, D [799-817]ORF 181 1450 CCAAGGCAGUGCUGAGCGA 2029 UCGCUCAGCACUGCCUUGG Rh [507-525]ORF 182 1451 GCAGAAGAAGGCUGUUGCA 2030 UGCAACAGCCUUCUUCUGC Rt [1120-1138]ORF 183 1452 GACAUUUUGUUGGAGCGUA 2031 UACGCUCCAACAAAAUGUC [2183-2201]3′UTR 184 1453 CGAGCACUCCAAGAUCAAA 2032 UUUGAUCUUGGAGUGCUCG Rh, D[697-715] ORF 185 1454 UCAUGAUGAUGCACCGGAA 2033 UUCCGGUGCAUCAUCAUGA Rh[930-948] ORF 186 1455 CCUGCUUCUCAGCGCCUUA 2034 UAAGGCGCUGAGAAGCAGG[241-259] ORF 187 1456 CCCAACCUCUCCCAACUAA 2035 UUAGUUGGGAGAGGUUGGG Rh[1895-1913] 3′UTR 188 1457 UGGGCAGACUCUGGUCAAA 2036 UUUGACCAGAGUCUGCCCARh [2007-2025] 3′UTR 189 1458 CUCUGGUCAAGAAGCAUCA 2037UGAUGCUUCUUGACCAGAG Rh [2015-2033] 3′UTR 190 1459 GAGCCUCUCGAGCGCCUUA2038 UAAGGCGCUCGAGAGGCUC [1055-1073] ORF 191 1460 AGAAGGCUGUUGCCAUCUA2039 UAGAUGGCAACAGCCUUCU Rt [1125-1143] ORF 192 1461 CCCUGCUAGUCAACGCCAA2040 UUGGCGUUGACUAGCAGGG Rh [822-840] ORF 193 1462 GCCUUCAGCUUGUACCAGA2041 UCUGGUACAAGCUGAAGGC [380-398] ORF 194 1463 GCUGCUAACCAAAGAGCAA 2042UUGCUCUUUGGUUAGCAGC [1078-1096] ORF 195 1464 CCCACAAGCUCUCCAGCCA 2043UGGCUGGAGAGCUUGUGGG Rh, D, P [1011-1029] ORF 196 1465GCUCCCUGCUAUUCAUUGA 2044 UCAAUGAAUAGCAGGGAGC D [1422-1440] ORF 197 1466GUUCUUCAAAGAUAGGGAA 2045 UUCCCUAUCUUUGAAGAAC [2083-2101] 3′UTR 198 1467GUCAGCCAGCCCUCUUCUA 2046 UAGAAGAGGGCUGGCUGAC Rh [1841-1859] 3′UTR 1991468 GCGGGACACCCAAAGCGGA 2047 UCCGCUUUGGGUGUCCCGC [1405-1423] ORF 2001469 AGCGCAGCGCGCUGCAGUA 2048 UACUGCAGCGCGCUGCGCU Rh, Rt [726-744] ORF201 1470 CCGGAAACUCCACAUCCUA 2049 UAGGAUGUGGAGUUUCCGG [1701-1719] 3′UTR202 1471 CCAUUGACAAGAACAAGGA 2050 UCCUUGUUCUUGUCAAUGG Rh, D [1215-1233]ORF 203 1472 GGACAUCUACGGGCGCGAA 2051 UUCGCGCCCGUAGAUGUCC D [1333-1351]ORF 204 1473 GACACAUGGGUGCUAUUGA 2052 UCAAUAGCACCCAUGUGUC Rh, Rt, M[1535-1553] 3′UTR 205 1474 CCUGGCACUGCGGAGAAGA 2053 UCUUCUCCGCAGUGCCAGG[317-335] ORF 206 1475 GGGCCUGACUGAGGCCAUA 2054 UAUGGCCUCAGUCAGGCCC Rt[1201-1219] ORF 207 1476 ACACUGGGAUGAGAAAUUA 2055 UAAUUUCUCAUCCCAGUGU Rh[853-871] ORF 208 1477 GGUCAGCCAGCCCUCUUCA 2056 UGAAGAGGGCUGGCUGACC Rh[1840-1858] 3′UTR 209 1478 GUGAGGCGGAUUGAGAAGA 2057 UCUUCUCAAUCCGCCUCAC[1969-1987] 3′UTR 210 1479 UCACCUGUGAGACCAAAUA 2058 UAUUUGGUCUCACAGGUGARh [1811-1829] 3′UTR 211 1480 AGCUGCAAAUCGUGGAGAA 2059UUCUCCACGAUUUGCAGCU Rh [984-1002] ORF 212 1481 GGUGCACACAGGAUGGCAA 2060UUGCCAUCCUGUGUGCACC Rh [1495-1513] 3′UTR 213 1482 GGGUGUGGUGGAGGUGACA2061 UGUCACCUCCACCACACCC Rh, D [1153-1171] ORF 214 1483CCAGCCUUGGAUACUCCAA 2062 UUGGAGUAUCCAAGGCUGG Rh [1575-1593] 3′UTR 2151484 CCACAAGCUCUCCAGCCUA 2063 UAGGCUGGAGAGCUUGUGG Rh, D, P [1012-1030]ORF 216 1485 AAAGGCUCCUGAGACACAA 2064 UUGUGUCUCAGGAGCCUUU [1523-1541]3′UTR 217 1486 AGGAAAAGCUGCAAAUCGA 2065 UCGAUUUGCAGCUUUUCCU Rh [978-996]ORF 218 1487 CGCAGCAGCUCCUGGCACA 2066 UGUGCCAGGAGCUGCUGCG [307-325] ORF219 1488 GGUGUCAUGAUGAUGCACA 2067 UGUGCAUCAUCAUGACACC Rh [926-944] ORF220 1489 CCUCUUCUGACACUAAAAA 2068 UUUUUAGUGUCAGAAGAGG [1851-1869] 3′UTR221 1490 AGCUAGAAUUCACUCCACA 2069 UGUGGAGUGAAUUCUAGCU Rh [1649-1667]3′UTR 222 1491 CGCUGGGCGGCAAGGCGAA 2070 UUCGCCUUGCCGCCCAGCG [474-492]ORF 223 1492 GGCCUGGCCUUCAGCUUGA 2071 UCAAGCUGAAGGCCAGGCC [374-392] ORF224 1493 AGACACAUGGGUGCUAUUA 2072 UAAUAGCACCCAUGUGUCU Rh, Rt, M[1534-1552] 3′UTR 225 1494 CGUGGGUGUCAUGAUGAUA 2073 UAUCAUCAUGACACCCACGRh [922-940] ORF 226 1495 GUGGGUGUCAUGAUGAUGA 2074 UCAUCAUCAUGACACCCACRh [923-941] ORF 227 1496 GAGAAGGAGCUCCCAGGAA 2075 UUCCUGGGAGCUCCUUCUC[1981-1999] 3′UTR 228 1497 GACUCUGGUCAAGAAGCAA 2076 UUGCUUCUUGACCAGAGUCRh [2013-2031] 3′UTR 229 1498 CACUAAAACACCUCAGCUA 2077UAGCUGAGGUGUUUUAGUG [1861-1879] 3′UTR 230 1499 GGAGGCAUCCAAAGGCUCA 2078UGAGCCUUUGGAUGCCUCC [1513-1531] 3′UTR 231 1500 GACCCAGCUCAGUGAGCUA 2079UAGCUCACUGAGCUGGGUC [636-654] ORF 232 1501 CCAUGACCUGCAGAAACAA 2080UUGUUUCUGCAGGUCAUGG Rh, Rt, M [1171-1189] ORF 233 1502AGAUGCAGAAGAAGGCUGA 2081 UCAGCCUUCUUCUGCAUCU Rh, Rt, M [1116-1134] ORF234 1503 CAGCAAGCAGCACUACAAA 2082 UUUGUAGUGCUGCUUGCUG Rh, D [676-694]ORF 235 1504 CAAGCUCUCCAGCCUCAUA 2083 UAUGAGGCUGGAGAGCUUG Rh, D, M, P[1015-1033] ORF 236 1505 UGCAGAAGAAGGCUGUUGA 2084 UCAACAGCCUUCUUCUGCA Rt[1119-1137] ORF 237 1506 GGCGCGAGGAGCUGCGCAA 2085 UUGCGCAGCUCCUCGCGCCRh, D, M [1344-1362] ORF 238 1507 GGUACCAGCCUUGGAUACA 2086UGUAUCCAAGGCUGGUACC Rh [1571-1589] 3′UTR 239 1508 GCAGCCGACUGUACGGACA2087 UGUCCGUACAGUCGGCUGC [621-639] ORF 240 1509 CAGCCUCAUCAUCCUCAUA 2088UAUGAGGAUGAUGAGGCUG Rh, D, Rt, M [1024-1042] ORF 241 1510GCCACCGCCUUUGAGUUGA 2089 UCAACUCAAAGGCGGUGGC Rh [1289-1307] ORF 242 1511AGAAGGACCUGUACCUGGA 2090 UCCAGGUACAGGUCCUUCU Rh, D [1257-1275] ORF 2431512 GGUGAAGAAACCUGCAGCA 2091 UGCUGCAGGUUUCUUCACC Rh [289-307] ORF 2441513 GUACCUUCUCACCUGUGAA 2092 UUCACAGGUGAGAAGGUAC Rh [1803-1821] 3′UTR245 1514 GGCCAAGGACCAGGCAGUA 2093 UACUGCCUGGUCCUUGGCC Rh [403-421] ORF246 1515 GGCGGCAAGGCGACCACGA 2094 UCGUGGUCGCCUUGCCGCC [479-497] ORF 2471516 AGCACUCCAAGAUCAACUA 2095 UAGUUGAUCUUGGAGUGCU Rh, D [699-717] ORF248 1517 AUAUUUAUAGCCAGGUACA 2096 UGUACCUGGCUAUAAAUAU Rh [1789-1807]3′UTR 249 1518 GGCAGCCGACUGUACGGAA 2097 UUCCGUACAGUCGGCUGCC [620-638]ORF 250 1519 GUCACGCAUGUCAGGCAAA 2098 UUUGCCUGACAUGCGUGAC Rh, D[1240-1258] ORF 251 1520 GACAGGCCUCUACAACUAA 2099 UUAGUUGUAGAGGCCUGUCRh, Rb, Rt, P [946-964] ORF 252 1521 GAUGCAGAAGAAGGCUGUA 2100UACAGCCUUCUUCUGCAUC Rh, Rt, M [1117-1135] ORF 253 1522ACCCAUGACCUGCAGAAAA 2101 UUUUCUGCAGGUCAUGGGU Rh, Rt, M [1169-1187] ORF254 1523 GGCUUCAUGGUGACUCGGA 2102 UCCGAGUCACCAUGAAGCC Rh [896-914] ORF255 1524 UGCCUCAAUCAGUAUUCAA 2103 UUGAAUACUGAUUGAGGCA [1772-1790] 3′UTR256 1525 GUUCUUCAAGCCACACUGA 2104 UCAGUGUGGCUUGAAGAAC Rh, Rb, D[841-859] ORF 257 1526 ACUCCAAGAUCAACUUCCA 2105 UGGAAGUUGAUCUUGGAGU Rh,D, Rt, M [702-720] ORF 258 1527 GCUGUUCUACGCCGACCAA 2106UUGGUCGGCGUAGAACAGC Rh [1369-1387] ORF 259 1528 UAGUCAACGCCAUGUUCUA 2107UAGAACAUGGCGUUGACUA Rh [828-846] ORF 260 1529 CCGUGUGCCUGAGCGGACA 2108UGUCCGCUCAGGCACACGG Rh [1625-1643] 3′UTR 261 1530 AGGCCUCUACAACUACUAA2109 UUAGUAGUUGUAGAGGCCU Rh, Rb, D, [949-967] Rt, P ORF 262 1531GCUUCAUGGUGACUCGGUA 2110 UACCGAGUCACCAUGAAGC Rh [897-915] ORF 263 1532GGUCAAGAAGCAUCGUGUA 2111 UACACGAUGCUUCUUGACC Rh [2019-2037] 3′UTR 2641533 CUGCGAGCACUCCAAGAUA 2112 UAUCUUGGAGUGCUCGCAG Rh, D [694-712] ORF265 1534 GUCCUAUACCGUGGGUGUA 2113 UACACCCACGGUAUAGGAC Rh [913-931] ORF266 1535 GGCCUGACUGAGGCCAUUA 2114 UAAUGGCCUCAGUCAGGCC Rh [1202-1220] ORF267 1536 CACUCCAAGAUCAACUUCA 2115 UGAAGUUGAUCUUGGAGUG Rh, D, Rt, M[701-719] ORF 268 1537 GCGUCGCAGGCCAAGGCAA 2116 UUGCCUUGGCCUGCGACGC[497-515] ORF 269 1538 AAGGGUGACAAGAUGCGAA 2117 UUCGCAUCUUGUCACCCUU Rh,D [1457-1475] ORF 270 1539 CAAGCUGUUCUACGCCGAA 2118 UUCGGCGUAGAACAGCUUGRh [1366-1384] ORF 271 1540 CCUGCUAGUCAACGCCAUA 2119 UAUGGCGUUGACUAGCAGGRh [823-841] ORF 272 1541 CCAAGGGUGUGGUGGAGGA 2120 UCCUCCACCACACCCUUGGRh, D [1149-1167] ORF 273 1542 CACACAGGAUGGCAGGAGA 2121UCUCCUGCCAUCCUGUGUG Rh [1499-1517] 3′UTR 274 1543 UCCUGAGACACAUGGGUGA2122 UCACCCAUGUGUCUCAGGA D, Rt, M [1529-1547] 3′UTR 275 1544CUACAACUACUACGACGAA 2123 UUCGUCGUAGUAGUUGUAG Rb [955-973] ORF 276 1545GACAAGAUGCGAGACGAGA 2124 UCUCGUCUCGCAUCUUGUC Rh, Rt [1463-1481] ORF 2771546 CCUGGAAGCUGGGCAGCCA 2125 UGGCUGCCCAGCUUCCAGG [609-627] ORF 278 1547CUUCAAGCCACACUGGGAA 2126 UUCCCAGUGUGGCUUGAAG Rh, Rb, D [844-862] ORF 2791548 GCGAGACGAGUUAUAGGGA 2127 UCCCUAUAACUCGUCUCGC Rh [1471-1489] ORF+ 3′UTR 280 1549 GAAGCUGGGCAGCCGACUA 2128 UAGUCGGCUGCCCAGCUUC [613-631]ORF 281 1550 GUGCCUGAGCGGACCUUCA 2129 UGAAGGUCCGCUCAGGCAC Rh [1629-1647]3′UTR 282 1551 GGUGACCCAUGACCUGCAA 2130 UUGCAGGUCAUGGGUCACC Rh, Rt, M[1165-1183] ORF 283 1552 AUGAGCCUUUGUUGCUAUA 2131 UAUAGCAACAAAGGCUCAU Rh[2114-2132] 3′UTR 284 1553 CAACUACUACGACGACGAA 2132 UUCGUCGUCGUAGUAGUUGRb [958-976] ORF 285 1554 GCUGCGCUCACUCAGCAAA 2133 UUUGCUGAGUGAGCGCAGCRh [571-589] ORF 286 1555 GAGAACAUCCUGGUGUCAA 2134 UUGACACCAGGAUGUUCUC[422-440] ORF 287 1556 CCCAAGCUGUUCUACGCCA 2135 UGGCGUAGAACAGCUUGGG Rh[1364-1382] ORF 288 1557 CAGCUCUAUCCCAACCUCA 2136 UGAGGUUGGGAUAGAGCUG[1886-1904] 3′UTR 289 1558 UGAGCUUCGCUGAUGACUA 2137 UAGUCAUCAGCGAAGCUCARh [648-666] ORF 290 1559 CCCAAGGCGGCCACGCUUA 2138 UAAGCGUGGCCGCCUUGGGRh [341-359] ORF 291 1560 CUAUACCGUGGGUGUCAUA 2139 UAUGACACCCACGGUAUAGRh [916-934] ORF 292 1561 CAUUGACAAGAACAAGGCA 2140 UGCCUUGUUCUUGUCAAUGRh, D [1216-1234] ORF 293 1562 GGACCCAGCUCAGUGAGCA 2141UGCUCACUGAGCUGGGUCC [635-653] ORF 294 1563 GACGACGAGAAGGAAAAGA 2142UCUUUUCCUUCUCGUCGUC Rh [968-986] ORF 295 1564 GCGGCAAGGCGACCACGGA 2143UCCGUGGUCGCCUUGCCGC [480-498] ORF 296 1565 GGGACACCCAAAGCGGCUA 2144UAGCCGCUUUGGGUGUCCC [1407-1425] ORF 297 1566 GGGAGGUGAGGUACCAGCA 2145UGCUGGUACCUCACCUCCC [1562-1580] 3′UTR 298 1567 GCAGCACUACAACUGCGAA 2146UUCGCAGUUGUAGUGCUGC Rh, D [682-700] ORF 299 1568 GCGCAACGUGACCUGGAAA2147 UUUCCAGGUCACGUUGCGC M [598-616] ORF 300 1569 GGGCUGGGCCUGACUGAGA2148 UCUCAGUCAGGCCCAGCCC [1196-1214] ORF 301 1570 CCUGAGCGGACCUUCCCAA2149 UUGGGAAGGUCCGCUCAGG Rh [1632-1650] 3′UTR 302 1571GCAGCUGAAGAUCUGGAUA 2150 UAUCCAGAUCUUCAGCUGC Rh, D [1093-1111] ORF 3031572 AGUGGAGAACAUCCUGGUA 2151 UACCAGGAUGUUCUCCACU Rh [418-436] ORF 3041573 GCAAGCAGCACUACAACUA 2152 UAGUUGUAGUGCUGCUUGC Rh, D [678-696] ORF305 1574 AGCUCAGUGAGCUUCGCUA 2153 UAGCGAAGCUCACUGAGCU [641-659] ORF 3061575 CCGACUUGUCACGCAUGUA 2154 UACAUGCGUGACAAGUCGG Rh [1233-1251] ORF 3071576 CCGAGGUCACCAAGGACGA 2155 UCGUCCUUGGUGACCUCGG Rh, D [786-804] ORF308 1577 GGAGCCUCUCGAGCGCCUA 2156 UAGGCGCUCGAGAGGCUCC [1054-1072] ORF309 1578 GGCCGCGCAGACCACCGAA 2157 UUCGGUGGUCUGCGCGGCC [757-775] ORF 3101579 GGAAACUCCACAUCCUGUA 2158 UACAGGAUGUGGAGUUUCC Rh [1703-1721] 3′UTR311 1580 CAAAGCGGCUCCCUGCUAA 2159 UUAGCAGGGAGCCGCUUUG [1415-1433] ORF312 1581 GCUCCUGAGACACAUGGGA 2160 UCCCAUGUGUCUCAGGAGC D [1527-1545]3′UTR 313 1582 CCUGGGCCAUAGUCAUUCA 2161 UGAAUGACUAUGGCCCAGG [1725-1743]3′UTR 314 1583 CGUGGAGCCUCUCGAGCGA 2162 UCGCUCGAGAGGCUCCACG [1051-1069]ORF 315 1584 CCUCCUGCUUCUCAGCGCA 2163 UGCGCUGAGAAGCAGGAGG [238-256] ORF316 1585 AGUCCCAGAUCAAGCCUGA 2164 UCAGGCUUGAUCUGGGACU Rh [1756-1774]3′UTR 317 1586 UACCGUGGGUGUCAUGAUA 2165 UAUCAUGACACCCACGGUA Rh [919-937]ORF 318 1587 GCCAGCCCUCUUCUGACAA 2166 UUGUCAGAAGAGGGCUGGC [1845-1863]3′UTR 319 1588 CCGAGGUGAAGAAACCUGA 2167 UCAGGUUUCUUCACCUCGG Rh, Rt[285-303] ORF 320 1589 UCCUGGCACUGCGGAGAAA 2168 UUUCUCCGCAGUGCCAGGA[316-334] ORF 321 1590 CCCGGAAACUCCACAUCCA 2169 UGGAUGUGGAGUUUCCGGG[1700-1718] 3′UTR 322 1591 ACUCUGGUCAAGAAGCAUA 2170 UAUGCUUCUUGACCAGAGURh [2014-2032] 3′UTR 323 1592 CCCAGAUACCAUGAUGCUA 2171UAGCAUCAUGGUAUCUGGG Rh [1679-1697] 3′UTR 324 1593 CCUGAGACACAUGGGUGCA2172 UGCACCCAUGUGUCUCAGG D, Rt, M [1530-1548] 3′UTR 325 1594GCACUACAACUGCGAGCAA 2173 UUGCUCGCAGUUGUAGUGC Rh, D [685-703] ORF 3261595 CCACAAGAUGGUGGACAAA 2174 UUUGUCCACCAUCUUGUGG Rh, Rb, M, P [874-892]ORF 327 1596 GGACACAGAUGGCAACCCA 2175 UGGGUUGCCAUCUGUGUCC [1306-1324]ORF 328 1597 GAAAAGCUGCUAACCAAAA 2176 UUUUGGUUAGCAGCUUUUC [1073-1091]ORF 329 1598 ACUACAACUGCGAGCACUA 2177 UAGUGCUCGCAGUUGUAGU Rh, D[687-705] ORF 330 1599 GCACUCCAAGAUCAACUUA 2178 UAAGUUGAUCUUGGAGUGC Rh,D [700-718] ORF 331 1600 GCCUUGAAAAGCUGCUAAA 2179 UUUAGCAGCUUUUCAAGGC[1068-1086] ORF 332 1601 GUGACUCGGUCCUAUACCA 2180 UGGUAUAGGACCGAGUCAC Rh[905-923] ORF 333 1602 GUGGUGGAGGUGACCCAUA 2181 UAUGGGUCACCUCCACCAC Rh,Rb, Rt, M [1157-1175] ORF 334 1603 AUGCGAGACGAGUUAUAGA 2182UCUAUAACUCGUCUCGCAU Rh [1469-1487] ORF + 3′UTR 335 1604ACCUUCCCAGCUAGAAUUA 2183 UAAUUCUAGCUGGGAAGGU Rh [1641-1659] 3′UTR 3361605 CCCAGCUAGAAUUCACUCA 2184 UGAGUGAAUUCUAGCUGGG Rh [1646-1664] 3′UTR337 1606 GGUCACCAAGGACGUGGAA 2185 UUCCACGUCCUUGGUGACC Rh, D [790-808]ORF 338 1607 GGCCUCAGGGUGCACACAA 2186 UUGUGUGCACCCUGAGGCC [1487-1505]3′UTR 339 1608 UGAGGUACCAGCCUUGGAA 2187 UUCCAAGGCUGGUACCUCA Rh[1568-1586] 3′UTR 340 1609 CAUGGUGACUCGGUCCUAA 2188 UUAGGACCGAGUCACCAUGRh [901-919] ORF 341 1610 GGUGAGGUACCAGCCUUGA 2189 UCAAGGCUGGUACCUCACCRh [1566-1584] 3′UTR 342 1611 GCCGAGGUGAAGAAACCUA 2190UAGGUUUCUUCACCUCGGC Rh, Rt [284-302] ORF 343 1612 GUACGGACCCAGCUCAGUA2191 UACUGAGCUGGGUCCGUAC [631-649] ORF 344 1613 CAAGAAGGACCUGUACCUA 2192UAGGUACAGGUCCUUCUUG Rh, D, M [1255-1273] ORF 345 1614GAGCACUCCAAGAUCAACA 2193 UGUUGAUCUUGGAGUGCUC Rh, D [698-716] ORF 3461615 CAUGUUCUUCAAGCCACAA 2194 UUGUGGCUUGAAGAACAUG Rh, Rb, D [838-856]ORF 347 1616 CCCUCCUGCUUCUCAGCGA 2195 UCGCUGAGAAGCAGGAGGG [237-255] ORF348 1617 AUGUCAGGCAAGAAGGACA 2196 UGUCCUUCUUGCCUGACAU Rh, D [1247-1265]ORF 349 1618 CAAGAUCAACUUCCGCGAA 2197 UUCGCGGAAGUUGAUCUUG D [706-724]ORF 350 1619 GCGUGUUCCACGCCACCGA 2198 UCGGUGGCGUGGAACACGC [1278-1296]ORF 351 1620 CGGACCCAGCUCAGUGAGA 2199 UCUCACUGAGCUGGGUCCG [634-652] ORF352 1621 CCUUCAGCUUGUACCAGGA 2200 UCCUGGUACAAGCUGAAGG [381-399] ORF 3531622 GCUCUCCAGCCUCAUCAUA 2201 UAUGAUGAGGCUGGAGAGC Rh, D, Rt, [1018-1036]M, P ORF 354 1623 CCCUGGCCCACAAGCUCUA 2202 UAGAGCUUGUGGGCCAGGG Rh, D, P[1005-1023] ORF 355 1624 GCCCGAGGUCACCAAGGAA 2203 UUCCUUGGUGACCUCGGGCRh, D [784-802] ORF 356 1625 GUGGAGAACAUCCUGGUGA 2204UCACCAGGAUGUUCUCCAC Rh [419-437] ORF 357 1626 GCUCACUCAGCAACUCCAA 2205UUGGAGUUGCUGAGUGAGC Rh [576-594] ORF 358 1627 ACGCCAUGUUCUUCAAGCA 2206UGCUUGAAGAACAUGGCGU Rh, Rb, P [834-852] ORF 359 1628 ACACAUGGGUGCUAUUGGA2207 UCCAAUAGCACCCAUGUGU Rh [1536-1554] 3′UTR 360 1629CCAGCUCAGUGAGCUUCGA 2208 UCGAAGCUCACUGAGCUGG [639-657] ORF 361 1630CCCAGCUCAGUGAGCUUCA 2209 UGAAGCUCACUGAGCUGGG [638-656] ORF 362 1631GGGCGGCAAGGCGACCACA 2210 UGUGGUCGCCUUGCCGCCC [478-496] ORF 363 1632CAGGGUGCACACAGGAUGA 2211 UCAUCCUGUGUGCACCCUG [1492-1510] 3′UTR 364 1633AGGUGAAGAAACCUGCAGA 2212 UCUGCAGGUUUCUUCACCU Rh [288-306] ORF 365 1634CCUCUCCCAACUAUAAAAA 2213 UUUUUAUAGUUGGGAGAGG Rh [1900-1918] 3′UTR 3661635 GACUGUACGGACCCAGCUA 2214 UAGCUGGGUCCGUACAGUC [627-645] ORF 367 1636GAAGGAGCUCCCAGGAGGA 2215 UCCUCCUGGGAGCUCCUUC [1983-2001] 3′UTR 368 1637ACGCAUGUCAGGCAAGAAA 2216 UUUCUUGCCUGACAUGCGU Rh, D [1243-1261] ORF 3691638 GACUCGGUCCUAUACCGUA 2217 UACGGUAUAGGACCGAGUC Rh [907-925] ORF 3701639 CACUACAACUGCGAGCACA 2218 UGUGCUCGCAGUUGUAGUG Rh, D [686-704] ORF371 1640 AGCUCCUGGCACUGCGGAA 2219 UUCCGCAGUGCCAGGAGCU [313-331] ORF 3721641 CUAAGGGUGACAAGAUGCA 2220 UGCAUCUUGUCACCCUUAG Rh [1455-1473] ORF 3731642 UGUGAGACCAAAUUGAGCA 2221 UGCUCAAUUUGGUCUCACA Rh [1816-1834] 3′UTR374 1643 GCCGACUUGUCACGCAUGA 2222 UCAUGCGUGACAAGUCGGC Rh [1232-1250] ORF375 1644 CAGGAUGGCAGGAGGCAUA 2223 UAUGCCUCCUGCCAUCCUG [1503-1521] 3′UTR376 1645 ACAAGAACAAGGCCGACUA 2224 UAGUCGGCCUUGUUCUUGU Rh [1221-1239] ORF377 1646 UGCGCUCCCUCCUGCUUCA 2225 UGAAGCAGGAGGGAGCGCA [231-249] ORF 3781647 GGCGAGCUGCUGCGCUCAA 2226 UUGAGCGCAGCAGCUCGCC Rh [563-581] ORF 3791648 GAUGCACCGGACAGGCCUA 2227 UAGGCCUGUCCGGUGCAUC Rh, Rb, Rt, [937-955]M, P ORF 380 1649 CGUGUCGCUGGGCGGCAAA 2228 UUUGCCGCCCAGCGACACG [469-487]ORF 381 1650 AUCCCAACCUCUCCCAACA 2229 UGUUGGGAGAGGUUGGGAU Rh [1893-1911]3′UTR 382 1651 UGUUCUACGCCGACCACCA 2230 UGGUGGUCGGCGUAGAACA Rh[1371-1389] ORF 383 1652 CGGCCUGGCCUUCAGCUUA 2231 UAAGCUGAAGGCCAGGCCG[373-391] ORF 384 1653 GUCGCAGGCCAAGGCAGUA 2232 UACUGCCUUGGCCUGCGAC[499-517] ORF 385 1654 AGUCAUUCUGCCUGCCCUA 2233 UAGGGCAGGCAGAAUGACU[1735-1753] 3′UTR 386 1655 CCCAGAAUGACCUGGCCGA 2234 UCGGCCAGGUCAUUCUGGG[1949-1967] 3′UTR 387 1656 ACAAGAUGGUGGACAACCA 2235 UGGUUGUCCACCAUCUUGURh, Rb, M, P [876-894] ORF 388 1657 GCUAGUCAACGCCAUGUUA 2236UAACAUGGCGUUGACUAGC Rh [826-844] ORF 389 1658 ACGCCACCGCCUUUGAGUA 2237UACUCAAAGGCGGUGGCGU Rh [1287-1305] ORF 390 1659 GCCGCGCAGACCACCGACA 2238UGUCGGUGGUCUGCGCGGC [758-776] ORF 391 1660 GCUAUUCAUUGGGCGCCUA 2239UAGGCGCCCAAUGAAUAGC D [1429-1447] ORF 392 1661 CUCAGUGAGCUUCGCUGAA 2240UUCAGCGAAGCUCACUGAG [643-661] ORF 393 1662 GGAGGUGAGGUACCAGCCA 2241UGGCUGGUACCUCACCUCC [1563-1581] 3′UTR 394 1663 GCCAAGGCAGUGCUGAGCA 2242UGCUCAGCACUGCCUUGGC Rh [506-524] ORF 395 1664 CUCUCCAGCCUCAUCAUCA 2243UGAUGAUGAGGCUGGAGAG Rh, D, Rt, [1019-1037] M, P ORF 396 1665GAAUGACCUGGCCGCAGUA 2244 UACUGCGGCCAGGUCAUUC [1953-1971] 3′UTR 397 1666UGGUGACUCGGUCCUAUAA 2245 UUAUAGGACCGAGUCACCA Rh [903-921] ORF 398 1667CAGGUACCUUCUCACCUGA 2246 UCAGGUGAGAAGGUACCUG Rh [1800-1818] 3′UTR 3991668 GUUCCACGCCACCGCCUUA 2247 UAAGGCGGUGGCGUGGAAC D [1282-1300] ORF 4001669 CCGACUGUACGGACCCAGA 2248 UCUGGGUCCGUACAGUCGG [625-643] ORF 401 1670GCAGACCACCGACGGCAAA 2249 UUUGCCGUCGGUGGUCUGC D, Rt [763-781] ORF 4021671 AAGAUGCGAGACGAGUUAA 2250 UUAACUCGUCUCGCAUCUU Rh [1466-1484] ORF 4031672 CAAAGAGCAGCUGAAGAUA 2251 UAUCUUCAGCUGCUCUUUG Rh [1087-1105] ORF 4041673 ACGACGAGAAGGAAAAGCA 2252 UGCUUUUCCUUCUCGUCGU Rh [969-987] ORF 4051674 CACUCCACUUGGACAUGGA 2253 UCCAUGUCCAAGUGGAGUG Rh [1659-1677] 3′UTR406 1675 AGUCCAUCAACGAGUGGGA 2254 UCCCACUCGUUGAUGGACU Rh, Rt, M[741-759] ORF 407 1676 GCGCCGGCCUGGCCUUCAA 2255 UUGAAGGCCAGGCCGGCGC Rh[369-387] ORF 408 1677 GGAAAAGCUGCAAAUCGUA 2256 UACGAUUUGCAGCUUUUCC Rh[979-997] ORF 409 1678 ACAUUUUGUUGGAGCGUGA 2257 UCACGCUCCAACAAAAUGU[2184-2202] 3′UTR 410 1679 ACCGUGGCUUCAUGGUGAA 2258 UUCACCAUGAAGCCACGGURh, Rt, M [891-909] ORF 411 1680 CCCUUCAUCUUCCUAGUGA 2259UCACUAGGAAGAUGAAGGG [1388-1406] ORF 412 1681 GAAAUUCCACCACAAGAUA 2260UAUCUUGUGGUGGAAUUUC Rh [865-883] ORF 413 1682 CUAUAAAACUAGGUGCUGA 2261UCAGCACCUAGUUUUAUAG Rh [1910-1928] 3′UTR 414 1683 GGAGGUGCACGCCGGCCUA2262 UAGGCCGGCGUGCACCUCC [544-562] ORF 415 1684 GCAGGCCAAGGCAGUGCUA 2263UAGCACUGCCUUGGCCUGC [502-520] ORF 416 1685 UGAGACCAAAUUGAGCUAA 2264UUAGCUCAAUUUGGUCUCA Rh [1818-1836] 3′UTR 417 1686 GCCAUAGUCAUUCUGCCUA2265 UAGGCAGAAUGACUAUGGC [1730-1748] 3′UTR 418 1687 AGCUGAAGAUCUGGAUGGA2266 UCCAUCCAGAUCUUCAGCU Rh, D [1095-1113] ORF 419 1688CCAUCUCCUUGCCCAAGGA 2267 UCCUUGGGCAAGGAGAUGG Rh [1137-1155] ORF 420 1689CCCAGAUCAAGCCUGCCUA 2268 UAGGCAGGCUUGAUCUGGG Rh [1759-1777] 3′UTR 4211690 GCUGUUGCCAUCUCCUUGA 2269 UCAAGGAGAUGGCAACAGC [1130-1148] ORF 4221691 CGAGGUCACCAAGGACGUA 2270 UACGUCCUUGGUGACCUCG Rh, D [787-805] ORF423 1692 CAACUAUAAAACUAGGUGA 2271 UCACCUAGUUUUAUAGUUG Rh [1907-1925]3′UTR 424 1693 GAAGGCUGUUGCCAUCUCA 2272 UGAGAUGGCAACAGCCUUC Rt[1126-1144] ORF 425 1694 UGCGGAGAAGUUGAGCCCA 2273 UGGGCUCAACUUCUCCGCA[325-343] ORF 426 1695 CUCCUUGCCCAAGGGUGUA 2274 UACACCCUUGGGCAAGGAG Rh[1141-1159] ORF 427 1696 GCCCUGAAAGUCCCAGAUA 2275 UAUCUGGGACUUUCAGGGC[1748-1766] 3′UTR 428 1697 CAAGGGUGUGGUGGAGGUA 2276 UACCUCCACCACACCCUUGRh, D [1150-1168] ORF 429 1698 AAGAGCAGCUGAAGAUCUA 2277UAGAUCUUCAGCUGCUCUU Rh [1089-1107] ORF 430 1699 GAAGAUGCAGAAGAAGGCA 2278UGCCUUCUUCUGCAUCUUC Rh, Rb, Rt [1114-1132] ORF 431 1700CGGAAACUCCACAUCCUGA 2279 UCAGGAUGUGGAGUUUCCG [1702-1720] 3′UTR 432 1701AGUCAACGCCAUGUUCUUA 2280 UAAGAACAUGGCGUUGACU Rh [829-847] ORF 433 1702CGAGCGCCUUGAAAAGCUA 2281 UAGCUUUUCAAGGCGCUCG [1063-1081] ORF 434 1703AUACCGUGGGUGUCAUGAA 2282 UUCAUGACACCCACGGUAU Rh [918-936] ORF 435 1704GACCUGGGCCAUAGUCAUA 2283 UAUGACUAUGGCCCAGGUC [1723-1741] 3′UTR 436 1705CAUGUCAGGCAAGAAGGAA 2284 UUCCUUCUUGCCUGACAUG Rh, D [1246-1264] ORF 4371706 UGCGAGACGAGUUAUAGGA 2285 UCCUAUAACUCGUCUCGCA Rh [1470-1488] ORF+ 3′UTR 438 1707 CGCAACGUGACCUGGAAGA 2286 UCUUCCAGGUCACGUUGCG [599-617]ORF 439 1708 AGCAAGCAGCACUACAACA 2287 UGUUGUAGUGCUGCUUGCU Rh, D[677-695] ORF 440 1709 GCUGCUGCGCUCACUCAGA 2288 UCUGAGUGAGCGCAGCAGC Rh[568-586] ORF 441 1710 UGAUGAUGCACCGGACAGA 2289 UCUGUCCGGUGCAUCAUCA Rh[933-951] ORF 442 1711 UUGUUGCUAUCAAUCCAAA 2290 UUUGGAUUGAUAGCAACAA Rh[2122-2140] 3′UTR 443 1712 CCUUGAAAAGCUGCUAACA 2291 UGUUAGCAGCUUUUCAAGG[1069-1087] ORF 444 1713 CCCUUUGACCAGGACAUCA 2292 UGAUGUCCUGGUCAAAGGGRh, Rt [1322-1340] ORF 445 1714 GAGGUGAAGAAACCUGCAA 2293UUGCAGGUUUCUUCACCUC Rh [287-305] ORF 446 1715 CCCAAGGGUGUGGUGGAGA 2294UCUCCACCACACCCUUGGG Rh, D [1148-1166] ORF 447 1716 CCCUGCUAUUCAUUGGGCA2295 UGCCCAAUGAAUAGCAGGG D [1425-1443] ORF 448 1717 CUGAAAGUCCCAGAUCAAA2296 UUUGAUCUGGGACUUUCAG [1751-1769] 3′UTR 449 1718 GCUGCAAAUCGUGGAGAUA2297 UAUCUCCACGAUUUGCAGC Rh [985-1003] ORF 450 1719 CAAGCCUGCCUCAAUCAGA2298 UCUGAUUGAGGCAGGCUUG Rh [1766-1784] 3′UTR 451 1720CGAGCAGCUGCGCGACGAA 2299 UUCGUCGCGCAGCUGCUCG [526-544] ORF 452 1721AGGCCGACUUGUCACGCAA 2300 UUGCGUGACAAGUCGGCCU Rh [1230-1248] ORF 453 1722GCAGCAGCUCCUGGCACUA 2301 UAGUGCCAGGAGCUGCUGC [308-326] ORF 454 1723GGCCAUAGUCAUUCUGCCA 2302 UGGCAGAAUGACUAUGGCC [1729-1747] 3′UTR 455 1724CCCGUGUGCCUGAGCGGAA 2303 UUCCGCUCAGGCACACGGG Rh [1624-1642] 3′UTR 4561725 CAGCUGAAGAUCUGGAUGA 2304 UCAUCCAGAUCUUCAGCUG Rh, D [1094-1112] ORF457 1726 CAAGCCACACUGGGAUGAA 2305 UUCAUCCCAGUGUGGCUUG Rh, Rb [847-865]ORF 458 1727 GAAUUCACUCCACUUGGAA 2306 UUCCAAGUGGAGUGAAUUC Rh [1654-1672]3′UTR 459 1728 CGGCGCCCUGCUAGUCAAA 2307 UUUGACUAGCAGGGCGCCG Rh [817-835]ORF 460 1729 UGGAAGCUGGGCAGCCGAA 2308 UUCGGCUGCCCAGCUUCCA [611-629] ORF461 1730 GGCAAGGCGACCACGGCGA 2309 UCGCCGUGGUCGCCUUGCC Rh [482-500] ORF462 1731 CACUGCGGAGAAGUUGAGA 2310 UCUCAACUUCUCCGCAGUG [322-340] ORF 4631732 GGCAGGAGGCAUCCAAAGA 2311 UCUUUGGAUGCCUCCUGCC [1509-1527] 3′UTR 4641733 GGUGACUCGGUCCUAUACA 2312 UGUAUAGGACCGAGUCACC Rh [904-922] ORF 4651734 UUUAUAGCCAGGUACCUUA 2313 UAAGGUACCUGGCUAUAAA Rh [1792-1810] 3′UTR466 1735 GGCCAUGGCCAAGGACCAA 2314 UUGGUCCUUGGCCAUGGCC Rh, D [397-415]ORF 467 1736 CAAAGAUAGGGAGGGAAGA 2315 UCUUCCCUCCCUAUCUUUG [2089-2107]3′UTR 468 1737 UCUUCUGACACUAAAACAA 2316 UUGUUUUAGUGUCAGAAGA [1853-1871]3′UTR 469 1738 CUUCUGACACUAAAACACA 2317 UGUGUUUUAGUGUCAGAAG [1854-1872]3′UTR 470 1739 UCACGUGGAGCCUCUCGAA 2318 UUCGAGAGGCUCCACGUGA [1048-1066]ORF 471 1740 CAGUCCAUCAACGAGUGGA 2319 UCCACUCGUUGAUGGACUG Rh, Rt, M[740-758] ORF 472 1741 AGACCAAAUUGAGCUAGGA 2320 UCCUAGCUCAAUUUGGUCU[1820-1838] 3′UTR 473 1742 GGGUUCCCGUGUGCCUGAA 2321 UUCAGGCACACGGGAACCCRh [1619-1637] 3′UTR 474 1743 UUGCUAUCAAUCCAAGAAA 2322UUUCUUGGAUUGAUAGCAA Rh [2125-2143] 3′UTR 475 1744 CAACCGUGGCUUCAUGGUA2323 UACCAUGAAGCCACGGUUG Rh, Rt, M [889-907] ORF 476 1745CUGUACGGACCCAGCUCAA 2324 UUGAGCUGGGUCCGUACAG [629-647] ORF 477 1746CAGCAGCAAGCAGCACUAA 2325 UUAGUGCUGCUUGCUGCUG Rh, D [673-691] ORF 4781747 CCUGCAGCCGCAGCAGCUA 2326 UAGCUGCUGCGGCUGCAGG [299-317] ORF 479 1748GACACUAAAACACCUCAGA 2327 UCUGAGGUGUUUUAGUGUC [1859-1877] 3′UTR 480 1749CAACUGCGAGCACUCCAAA 2328 UUUGGAGUGCUCGCAGUUG Rh, D [691-709] ORF 4811750 ACUGCGGAGAAGUUGAGCA 2329 UGCUCAACUUCUCCGCAGU [323-341] ORF 482 1751GCGCCCUGCUAGUCAACGA 2330 UCGUUGACUAGCAGGGCGC Rh [819-837] ORF 483 1752GGAAGCUGGGCAGCCGACA 2331 UGUCGGCUGCCCAGCUUCC [612-630] ORF 484 1753AGGCUCCUGAGACACAUGA 2332 UCAUGUGUCUCAGGAGCCU D [1525-1543] 3′UTR 4851754 CGACAAGCGCAGCGCGCUA 2333 UAGCGCGCUGCGCUUGUCG [721-739] ORF 486 1755UCAGUGAGCUUCGCUGAUA 2334 UAUCAGCGAAGCUCACUGA [644-662] ORF 487 1756UUGAGAAGGAGCUCCCAGA 2335 UCUGGGAGCUCCUUCUCAA [1979-1997] 3′UTR 488 1757ACUGCGAGCACUCCAAGAA 2336 UUCUUGGAGUGCUCGCAGU Rh, D [693-711] ORF 4891758 CAUCCUGGUGUCACCCGUA 2337 UACGGGUGACACCAGGAUG [427-445] ORF 490 1759GUGCGCAGCAGCAAGCAGA 2338 UCUGCUUGCUGCUGCGCAC Rh, D [668-686] ORF 4911760 CACGCCACCGCCUUUGAGA 2339 UCUCAAAGGCGGUGGCGUG Rh [1286-1304] ORF 4921761 UCUCGAGCGCCUUGAAAAA 2340 UUUUUCAAGGCGCUCGAGA [1060-1078] ORF 4931762 GCUUCGCUGAUGACUUCGA 2341 UCGAAGUCAUCAGCGAAGC Rh [651-669] ORF 4941763 UCUCCUUGCCCAAGGGUGA 2342 UCACCCUUGGGCAAGGAGA Rh [1140-1158] ORF 4951764 GCAGUCCAUCAACGAGUGA 2343 UCACUCGUUGAUGGACUGC Rh, Rt, M [739-757]ORF 496 1765 AGAUGGUGGACAACCGUGA 2344 UCACGGUUGUCCACCAUCU Rh, M[879-897] ORF 497 1766 CGGCUCCCUGCUAUUCAUA 2345 UAUGAAUAGCAGGGAGCCG[1420-1438] ORF 498 1767 AUACCAUGAUGCUGAGCCA 2346 UGGCUCAGCAUCAUGGUAU[1684-1702] 3′UTR 499 1768 AGCCAGGUACCUUCUCACA 2347 UGUGAGAAGGUACCUGGCURh [1797-1815] 3′UTR 500 1769 GAGCCCGGAAACUCCACAA 2348UUGUGGAGUUUCCGGGCUC [1697-1715] 3′UTR 501 1770 GCAGCUCCUGGCACUGCGA 2349UCGCAGUGCCAGGAGCUGC [311-329] ORF 502 1771 CCCGAGGUCACCAAGGACA 2350UGUCCUUGGUGACCUCGGG Rh, D [785-803] ORF 503 1772 CCUGACUGAGGCCAUUGAA2351 UUCAAUGGCCUCAGUCAGG Rh [1204-1222] ORF 504 1773 UGCUGAGCCCGGAAACUCA2352 UGAGUUUCCGGGCUCAGCA [1693-1711] 3′UTR 505 1774 GCCAUCUCCUUGCCCAAGA2353 UCUUGGGCAAGGAGAUGGC Rh [1136-1154] ORF 506 1775 CAAGCAGCACUACAACUGA2354 UCAGUUGUAGUGCUGCUUG Rh, D [679-697] ORF 507 1776CAAGGCAGUGCUGAGCGCA 2355 UGCGCUCAGCACUGCCUUG Rh [508-526] ORF 508 1777CAAUGACAUUUUGUUGGAA 2356 UUCCAACAAAAUGUCAUUG [2179-2197] 3′UTR 509 1778AGUGAGCUUCGCUGAUGAA 2357 UUCAUCAGCGAAGCUCACU [646-664] ORF 510 1779AUGAUGAUGCACCGGACAA 2358 UUGUCCGGUGCAUCAUCAU Rh [932-950] ORF 511 1780GAAACACCUGGCUGGGCUA 2359 UAGCCCAGCCAGGUGUUUC D [1183-1201] ORF 512 1781CCUGCUAUUCAUUGGGCGA 2360 UCGCCCAAUGAAUAGCAGG D [1426-1444] ORF 513 1782CGCCACCGCCUUUGAGUUA 2361 UAACUCAAAGGCGGUGGCG Rh [1288-1306] ORF 514 1783GCUUCUCAGCGCCUUCUGA 2362 UCAGAAGGCGCUGAGAAGC [244-262] ORF 515 1784UGAUGCUGAGCCCGGAAAA 2363 UUUUCCGGGCUCAGCAUCA [1690-1708] 3′UTR 516 1785UGACCUGGCCGCAGUGAGA 2364 UCUCACUGCGGCCAGGUCA [1956-1974] 3′UTR 517 1786UGCAGAAACACCUGGCUGA 2365 UCAGCCAGGUGUUUCUGCA [1179-1197] ORF 518 1787GCAGUGCUGAGCGCCGAGA 2366 UCUCGGCGCUCAGCACUGC [512-530] ORF 519 1788CGGCGCGCAACGUGACCUA 2367 UAGGUCACGUUGCGCGCCG [594-612] ORF 520 1789AGUGCUGAGCGCCGAGCAA 2368 UUGCUCGGCGCUCAGCACU [514-532] ORF 521 1790ACAGGCCUCUACAACUACA 2369 UGUAGUUGUAGAGGCCUGU Rh, Rb, D, [947-965] Rt, PORF 522 1791 GCAGCUGCGCGACGAGGAA 2370 UUCCUCGUCGCGCAGCUGC Rh, D[529-547] ORF 523 1792 AUUGAGAAGGAGCUCCCAA 2371 UUGGGAGCUCCUUCUCAAU[1978-1996] 3′UTR 524 1793 CGCGCAGACCACCGACGGA 2372 UCCGUCGGUGGUCUGCGCG[760-778] ORF 525 1794 CCUGUACCUGGCCAGCGUA 2373 UACGCUGGCCAGGUACAGG Rh[1264-1282] ORF 526 1795 CUGAGCGGACCUUCCCAGA 2374 UCUGGGAAGGUCCGCUCAG Rh[1633-1651] 3′UTR 527 1796 GGCCUUCAGCUUGUACCAA 2375 UUGGUACAAGCUGAAGGCC[379-397] ORF 528 1797 CACCCAAAGCGGCUCCCUA 2376 UAGGGAGCCGCUUUGGGUG[1411-1429] ORF 529 1798 GCCAAGGACCAGGCAGUGA 2377 UCACUGCCUGGUCCUUGGC Rh[404-422] ORF 530 1799 CUCAGGGUGCACACAGGAA 2378 UUCCUGUGUGCACCCUGAG[1490-1508] 3′UTR 531 1800 CGAGCUGCUGCGCUCACUA 2379 UAGUGAGCGCAGCAGCUCGRh [565-583] ORF 532 1801 GGCUGGGCCUGACUGAGGA 2380 UCCUCAGUCAGGCCCAGCC[1197-1215] ORF 533 1802 CCGCAGCAGCUCCUGGCAA 2381 UUGCCAGGAGCUGCUGCGG[306-324] ORF 534 1803 UGUGGGACCUGGGCCAUAA 2382 UUAUGGCCCAGGUCCCACA[1718-1736] 3′UTR 535 1804 AAGAUGCAGAAGAAGGCUA 2383 UAGCCUUCUUCUGCAUCUURh, Rt, M [1115-1133] ORF 536 1805 CCACGGCGCGCAACGUGAA 2384UUCACGUUGCGCGCCGUGG Rh [591-609] ORF 537 1806 ACCUUCUCACCUGUGAGAA 2385UUCUCACAGGUGAGAAGGU Rh [1805-1823] 3′UTR 538 1807 UGAAGAAACCUGCAGCCGA2386 UCGGCUGCAGGUUUCUUCA [291-309] ORF 539 1808 CAGCACUACAACUGCGAGA 2387UCUCGCAGUUGUAGUGCUG Rh, D [683-701] ORF 540 1809 GCGACAAGCGCAGCGCGCA2388 UGCGCGCUGCGCUUGUCGC [720-738] ORF 541 1810 UAGAAUUCACUCCACUUGA 2389UCAAGUGGAGUGAAUUCUA Rh [1652-1670] 3′UTR 542 1811 GUGGAAAAACAGACCGGGA2390 UCCCGGUCUGUUUUUCCAC [1603-1621] 3′UTR 543 1812 ACGUGGAGCCUCUCGAGCA2391 UGCUCGAGAGGCUCCACGU [1050-1068] ORF 544 1813 GGCGCGCAACGUGACCUGA2392 UCAGGUCACGUUGCGCGCC [595-613] ORF 545 1814 UGGACAACCGUGGCUUCAA 2393UUGAAGCCACGGUUGUCCA Rh, M [885-903] ORF 546 1815 CUAGUCAACGCCAUGUUCA2394 UGAACAUGGCGUUGACUAG Rh [827-845] ORF 547 1816 AGAAUGACCUGGCCGCAGA2395 UCUGCGGCCAGGUCAUUCU [1952-1970] 3′UTR 548 1817 AGCUGCUGCGCUCACUCAA2396 UUGAGUGAGCGCAGCAGCU Rh [567-585] ORF 549 1818 CUCUAUCCCAACCUCUCCA2397 UGGAGAGGUUGGGAUAGAG Rh [1889-1907] 3′UTR 550 1819GCGAGCUGCUGCGCUCACA 2398 UGUGAGCGCAGCAGCUCGC Rh [564-582] ORF 551 1820CGCAGCAGCAAGCAGCACA 2399 UGUGCUGCUUGCUGCUGCG Rh, D [671-689] ORF 5521821 GGCUGGGCUGGGCCUGACA 2400 UGUCAGGCCCAGCCCAGCC [1192-1210] ORF 5531822 UCUCCAGCCUCAUCAUCCA 2401 UGGAUGAUGAGGCUGGAGA Rh, D, Rt, M[1020-1038] ORF 554 1823 CAACGCCAUGUUCUUCAAA 2402 UUUGAAGAACAUGGCGUUGRh, Rb, P [832-850] ORF 555 1824 UGGCACUGCGGAGAAGUUA 2403UAACUUCUCCGCAGUGCCA [319-337] ORF 556 1825 UUUGAGUUGGACACAGAUA 2404UAUCUGUGUCCAACUCAAA [1298-1316] ORF 557 1826 UGGGCGAGCUGCUGCGCUA 2405UAGCGCAGCAGCUCGCCCA Rh [561-579] ORF 558 1827 CUGCUAACCAAAGAGCAGA 2406UCUGCUCUUUGGUUAGCAG [1079-1097] ORF 559 1828 AACGUGACCUGGAAGCUGA 2407UCAGCUUCCAGGUCACGUU [602-620] ORF 560 1829 AUGACAUUUUGUUGGAGCA 2408UGCUCCAACAAAAUGUCAU [2181-2199] 3′UTR 561 1830 CAGGAGGCAUCCAAAGGCA 2409UGCCUUUGGAUGCCUCCUG [1511-1529] 3′UTR 562 1831 AUCUCCUUGCCCAAGGGUA 2410UACCCUUGGGCAAGGAGAU Rh [1139-1157] ORF 563 1832 UGGGAUGAGAAAUUCCACA 2411UGUGGAAUUUCUCAUCCCA Rh [857-875] ORF 564 1833 AAAGCUGCUAACCAAAGAA 2412UUCUUUGGUUAGCAGCUUU [1075-1093] ORF 565 1834 AGGAGGCAUCCAAAGGCUA 2413UAGCCUUUGGAUGCCUCCU [1512-1530] 3′UTR 566 1835 CACCGCCUUUGAGUUGGAA 2414UUCCAACUCAAAGGCGGUG Rh [1291-1309] ORF 567 1836 CCAACUAUAAAACUAGGUA 2415UACCUAGUUUUAUAGUUGG Rh [1906-1924] 3′UTR 568 1837 CAAGAAGCAUCGUGUCUGA2416 UCAGACACGAUGCUUCUUG Rh [2022-2040] 3′UTR 569 1838AGCAGCUGAAGAUCUGGAA 2417 UUCCAGAUCUUCAGCUGCU Rh, D [1092-1110] ORF 5701839 GCGCUCCCUCCUGCUUCUA 2418 UAGAAGCAGGAGGGAGCGC [232-250] ORF 571 1840UGCUAGUCAACGCCAUGUA 2419 UACAUGGCGUUGACUAGCA Rh [825-843] ORF 572 1841CGCCGAGCAGCUGCGCGAA 2420 UUCGCGCAGCUGCUCGGCG [523-541] ORF 573 1842CCGCGCAGACCACCGACGA 2421 UCGUCGGUGGUCUGCGCGG [759-777] ORF 574 1843UAGCCAGGUACCUUCUCAA 2422 UUGAGAAGGUACCUGGCUA Rh [1796-1814] 3′UTR 5751844 UGCUUCUCAGCGCCUUCUA 2423 UAGAAGGCGCUGAGAAGCA [243-261] ORF 576 1845CUCCCUCCUGCUUCUCAGA 2424 UCUGAGAAGCAGGAGGGAG [235-253] ORF 577 1846CGCAGGCCAAGGCAGUGCA 2425 UGCACUGCCUUGGCCUGCG [501-519] ORF 578 1847GCAAGGCGACCACGGCGUA 2426 UACGCCGUGGUCGCCUUGC Rh [483-501] ORF 579 1848GCAGCCGCAGCAGCUCCUA 2427 UAGGAGCUGCUGCGGCUGC [302-320] ORF

TABLE E SERPINH1 Cross-Species 18 + 1-mer siRNAs human- SEQ Other32454740 No. SEQ ID NO Sense siRNA ID NO AntiSense siRNA SpeciesORF: 230-1486 1 2428 UCACCAAGGACGUGGAGCA 2576 UGCUCCACGUCCUUGGU Rh, D[792-810] GA ORF 2 2429 CAGCGCGCUGCAGUCCAUA 2577 UAUGGACUGCAGCGCGCRh, Rt [730-748] UG ORF 3 2430 CAUCUACGGGCGCGAGGAA 2578UUCCUCGCGCCCGUAGA D, M [1336-1354] UG ORF 4 2431 CUCCAGCCUCAUCAUCCUA2579 UAGGAUGAUGAGGCUGG Rh, D, Rt, M [1021-1039] AG ORF 5 2432GACAUCUACGGGCGCGAGA 2580 UCUCGCGCCCGUAGAUG D, M [1334-1352] UC ORF 62433 CGUGCGCAGCAGCAAGCAA 2581 UUGCUUGCUGCUGCGCA Rh, D, M [667-685] CGORF 7 2434 GUCACCAAGGACGUGGAGA 2582 UCUCCACGUCCUUGGUG Rh, D [791-809] ACORF 8 2435 CCGCGACAAGCGCAGCGCA 2583 UGCGCUGCGCUUGUCGC D [718-736] GG ORF9 2436 GCGCAGCGCGCUGCAGUCA 2584 UGACUGCAGCGCGCUGC Rh, Rt [727-745] GCORF 10 2437 GGCCCACAAGCUCUCCAGA 2585 UCUGGAGAGCUUGUGGG Rh, D, P[1009-1027] CC ORF 11 2438 CAAGGACGUGGAGCGCACA 2586 UGUGCGCUCCACGUCCURh, D [796-814] UG ORF 12 2439 AGCCUCAUCAUCCUCAUGA 2587UCAUGAGGAUGAUGAGG Rh, D, Rt, M [1025-1043] CU ORF 13 2440GGUGUGGUGGAGGUGACCA 2588 UGGUCACCUCCACCACA Rh, D [1154-1172] CC ORF 142441 GCAAGCUGCCCGAGGUCAA 2589 UUGACCUCGGGCAGCUU Rh, D [777-795] GC ORF15 2442 GUGGAGGUGACCCAUGACA 2590 UGUCAUGGGUCACCUCC Rh, Rt, M [1160-1178]AC ORF 16 2443 CACAAGAUGGUGGACAACA 2591 UGUUGUCCACCAUCUUG Rh, Rb, M, P[875-893] UG ORF 17 2444 GCGAGGAGCUGCGCAGCCA 2592 UGGCUGCGCAGCUCCUC D, M[1347-1365] GC ORF 18 2445 UACUACGACGACGAGAAGA 2593 UCUUCUCGUCGUCGUAG Rb[962-980] UA ORF 19 2446 GAGGUGACCCAUGACCUGA 2594 UCAGGUCAUGGGUCACCRh, Rt, M [1163-1181] UC ORF 20 2447 ACUUCCGCGACAAGCGCAA 2595UUGCGCUUGUCGCGGAA D [714-732] GU ORF 21 2448 GCCCACAAGCUCUCCAGCA 2596UGCUGGAGAGCUUGUGG Rh, D, P [1010-1028] GC ORF 22 2449GCGCAGCAGCAAGCAGCAA 2597 UUGCUGCUUGCUGCUGC Rh, D [670-688] GC ORF 232450 CGAGGAGCUGCGCAGCCCA 2598 UGGGCUGCGCAGCUCCU D, M [1348-1366] CG ORF24 2451 AACGCCAUGUUCUUCAAGA 2599 UCUUGAAGAACAUGGCG Rh, Rb, P [833-851]UU ORF 25 2452 GUCAGGCAAGAAGGACCUA 2600 UAGGUCCUUCUUGCCUG Rh, D[1249-1267] AC ORF 26 2453 GCCUGGGCGAGCUGCUGCA 2601 UGCAGCAGCUCGCCCAGRh, D [558-576] GC ORF 27 2454 GAUGAUGCACCGGACAGGA 2602UCCUGUCCGGUGCAUCA Rh, Rb, Rt, M [934-952] UC ORF 28 2455GGACCUGUACCUGGCCAGA 2603 UCUGGCCAGGUACAGGU Rh, D [1261-1279] CC ORF 292456 GCGACGAGGAGGUGCACGA 2604 UCGUGCACCUCCUCGUC D [537-555] GC ORF 302457 UGUGGUGGAGGUGACCCAA 2605 UUGGGUCACCUCCACCA Rh, D [1156-1174] CA ORF31 2458 UUCAAGCCACACUGGGAUA 2606 UAUCCCAGUGUGGCUUG Rh, Rb [845-863] AAORF 32 2459 CAAGAUGGUGGACAACCGA 2607 UCGGUUGUCCACCAUCU Rh, Rb, M, P[877-895] UG ORF 33 2460 UCAACUUCCGCGACAAGCA 2608 UGCUUGUCGCGGAAGUU D[711-729] GA ORF 34 2461 AUUCAUUGGGCGCCUGGUA 2609 UACCAGGCGCCCAAUGA D[1432-1450] AU ORF 35 2462 CUCCAAGAUCAACUUCCGA 2610 UCGGAAGUUGAUCUUGGRh, D, Rt, M [703-721] AG ORF 36 2463 CAGGCCAUGGCCAAGGACA 2611UGUCCUUGGCCAUGGCC Rh, D [395-413] UG ORF 37 2464 GUACCAGGCCAUGGCCAAA2612 UUUGGCCAUGGCCUGGU Rh, D [391-409] AC ORF 38 2465UGUCAGGCAAGAAGGACCA 2613 UGGUCCUUCUUGCCUGA Rh, D [1248-1266] CA ORF 392466 CUUCGUGCGCAGCAGCAAA 2614 UUUGCUGCUGCGCACGA Rh, D, M [664-682] AGORF 40 2467 CAACUUCCGCGACAAGCGA 2615 UCGCUUGUCGCGGAAGU D [712-730] UGORF 41 2468 CCACCACAAGAUGGUGGAA 2616 UUCCACCAUCUUGUGGU Rh, Rb, D, P[871-889] GG ORF 42 2469 GCGCGACGAGGAGGUGCAA 2617 UUGCACCUCCUCGUCGCRh, D [535-553] GC ORF 43 2470 CUACAACUGCGAGCACUCA 2618UGAGUGCUCGCAGUUGU Rh, D [688-706] AG ORF 44 2471 UGGAGGUGACCCAUGACCA2619 UGGUCAUGGGUCACCUC Rh, Rt, M [1161-1179] CA ORF 45 2472GAGGUCACCAAGGACGUGA 2620 UCACGUCCUUGGUGACC Rh, D [788-806] UC ORF 462473 AAGAAGGACCUGUACCUGA 2621 UCAGGUACAGGUCCUUC Rh, D [1256-1274] UU ORF47 2474 GACAACCGUGGCUUCAUGA 2622 UCAUGAAGCCACGGUUG Rh, Rt, M [887-905]UC ORF 48 2475 ACCAGGACAUCUACGGGCA 2623 UGCCCGUAGAUGUCCUG D, Rt[1329-1347] GU ORF 49 2476 GCUGCCCGAGGUCACCAAA 2624 UUUGGUGACCUCGGGCARh, D [781-799] GC ORF 50 2477 AUGCAGAAGAAGGCUGUUA 2625UAACAGCCUUCUUCUGC Rt [1118-1136] AU ORF 51 2478 GGCCUGGGCGAGCUGCUGA 2626UCAGCAGCUCGCCCAGG Rh, D [557-575] CC ORF 52 2479 GAUGGUGGACAACCGUGGA2627 UCCACGGUUGUCCACCA Rh, M [880-898] UC ORF 53 2480CUCCCUGCUAUUCAUUGGA 2628 UCCAAUGAAUAGCAGGG D [1423-1441] AG ORF 54 2481GAAGGACCUGUACCUGGCA 2629 UGCCAGGUACAGGUCCU Rh, D [1258-1276] UC ORF 552482 CCACCGACGGCAAGCUGCA 2630 UGCAGCUUGCCGUCGGU D, Rt [768-786] GG ORF56 2483 UGCUAUUCAUUGGGCGCCA 2631 UGGCGCCCAAUGAAUAG D [1428-1446] CA ORF57 2484 AUGUUCUUCAAGCCACACA 2632 UGUGUGGCUUGAAGAAC Rh, Rb, D [839-857]AU ORF 58 2485 CCAGGACAUCUACGGGCGA 2633 UCGCCCGUAGAUGUCCU D, Rt[1330-1348] GG ORF 59 2486 GCGCGAGGAGCUGCGCAGA 2634 UCUGCGCAGCUCCUCGCRh, D, M [1345-1363] GC ORF 60 2487 GAGCAGCUGCGCGACGAGA 2635UCUCGUCGCGCAGCUGC Rh, D [527-545] UC ORF 61 2488 CUAUUCAUUGGGCGCCUGA2636 UCAGGCGCCCAAUGAAU D [1430-1448] AG ORF 62 2489 ACAAGCUCUCCAGCCUCAA2637 UUGAGGCUGGAGAGCUU Rh, D, M, P [1014-1032] GU ORF 63 2490GCUGAAGAUCUGGAUGGGA 2638 UCCCAUCCAGAUCUUCA Rh, D [1096-1114] GC ORF 642491 GACCAGGACAUCUACGGGA 2639 UCCCGUAGAUGUCCUGG D, Rt [1328-1346] UC ORF65 2492 CAAGCGCAGCGCGCUGCAA 2640 UUGCAGCGCGCUGCGCU Rh, Rt [724-742] UGORF 66 2493 CCAUGGCCAAGGACCAGGA 2641 UCCUGGUCCUUGGCCAU Rh, D [399-417]GG ORF 67 2494 CACCAAGGACGUGGAGCGA 2642 UCGCUCCACGUCCUUGG Rh, D[793-811] UG ORF 68 2495 CCGUGGCUUCAUGGUGACA 2643 UGUCACCAUGAAGCCACRh, Rt, M [892-910] GG ORF 69 2496 UGACCAGGACAUCUACGGA 2644UCCGUAGAUGUCCUGGU Rt [1327-1345] CA ORF 70 2497 AGACCACCGACGGCAAGCA 2645UGCUUGCCGUCGGUGGU D, Rt [765-783] CU ORF 71 2498 GACAAGCGCAGCGCGCUGA2646 UCAGCGCGCUGCGCUUG Rh, Rt [722-740] UC ORF 72 2499AGAAACACCUGGCUGGGCA 2647 UGCCCAGCCAGGUGUUU D [1182-1200] CU ORF 73 2500AAGAUGGUGGACAACCGUA 2648 UACGGUUGUCCACCAUC Rh, M [878-896] UU ORF 742501 CAGACCACCGACGGCAAGA 2649 UCUUGCCGUCGGUGGUC D, Rt [764-782] UG ORF75 2502 AGGACCUGUACCUGGCCAA 2650 UUGGCCAGGUACAGGUC Rh, D [1260-1278] CUORF 76 2503 CUGCUAUUCAUUGGGCGCA 2651 UGCGCCCAAUGAAUAGC D [1427-1445] AGORF 77 2504 GUCCAUCAACGAGUGGGCA 2652 UGCCCACUCGUUGAUGG Rh, Rt, M[742-760] AC ORF 78 2505 CCAGGCCAUGGCCAAGGAA 2653 UUCCUUGGCCAUGGCCURh, D [394-412] GG ORF 79 2506 AAGCAGCACUACAACUGCA 2654UGCAGUUGUAGUGCUGC Rh, D [680-698] UU ORF 80 2507 UGUUCCACGCCACCGCCUA2655 UAGGCGGUGGCGUGGAA D [1281-1299] CA ORF 81 2508 UACAACUACUACGACGACA2656 UGUCGUCGUAGUAGUUG Rb [956-974] UA ORF 82 2509 CCUCAUCAUCCUCAUGCCA2657 UGGCAUGAGGAUGAUGA Rh, D, Rt, M [1027-1045] GG ORF 83 2510UGGUGGACAACCGUGGCUA 2658 UAGCCACGGUUGUCCAC Rh, M [882-900] CA ORF 842511 GACCACCGACGGCAAGCUA 2659 UAGCUUGCCGUCGGUGG D, Rt [766-784] UC ORF85 2512 AGCUGCGCGACGAGGAGGA 2660 UCCUCCUCGUCGCGCAG Rh, D [531-549] CUORF 86 2513 CGGCAAGCUGCCCGAGGUA 2661 UACCUCGGGCAGCUUGC Rh, D [775-793]CG ORF 87 2514 UGGCCCACAAGCUCUCCAA 2662 UUGGAGAGCUUGUGGGC Rh, D, P[1008-1026] CA ORF 88 2515 CAGCUGCGCGACGAGGAGA 2663 UCUCCUCGUCGCGCAGCRh, D [530-548] UG ORF 89 2516 CUUCCGCGACAAGCGCAGA 2664UCUGCGCUUGUCGCGGA D [715-733] AG ORF 90 2517 UGGGCCUGACUGAGGCCAA 2665UUGGCCUCAGUCAGGCC Rt [1200-1218] CA ORF 91 2518 GCUGCGCGACGAGGAGGUA 2666UACCUCCUCGUCGCGCA Rh, D [532-550] GC ORF 92 2519 CAGGACAUCUACGGGCGCA2667 UGCGCCCGUAGAUGUCC D [1331-1349] UG ORF 93 2520 GCCAUGGCCAAGGACCAGA2668 UCUGGUCCUUGGCCAUG Rh, D [398-416] GC ORF 94 2521UCCAAGAUCAACUUCCGCA 2669 UGCGGAAGUUGAUCUUG D [704-722] GA ORF 95 2522ACCACCGACGGCAAGCUGA 2670 UCAGCUUGCCGUCGGUG D, Rt [767-785] GU ORF 962523 AUCUACGGGCGCGAGGAGA 2671 UCUCCUCGCGCCCGUAG D, M [1337-1355] AU ORF97 2524 CUGCCCGAGGUCACCAAGA 2672 UCUUGGUGACCUCGGGC Rh, D [782-800] AGORF 98 2525 AUCAACUUCCGCGACAAGA 2673 UCUUGUCGCGGAAGUUG D [710-728] AUORF 99 2526 UCAUUGGGCGCCUGGUCCA 2674 UGGACCAGGCGCCCAAU Rh, D [1434-1452]GA ORF 100 2527 CAUUGGGCGCCUGGUCCGA 2675 UCGGACCAGGCGCCCAA Rh, D[1435-1453] UG ORF 101 2528 GUGUUCCACGCCACCGCCA 2676 UGGCGGUGGCGUGGAAC D[1280-1298] AC ORF 102 2529 AUGAUGCACCGGACAGGCA 2677 UGCCUGUCCGGUGCAUCRh, Rb, Rt, M, P [935-953] AU ORF 103 2530 CGACGAGGAGGUGCACGCA 2678UGCGUGCACCUCCUCGU D [538-556] CG ORF 104 2531 CAGAAACACCUGGCUGGGA 2679UCCCAGCCAGGUGUUUC D [1181-1199] UG ORF 105 2532 UGAUGCACCGGACAGGCCA 2680UGGCCUGUCCGGUGCAU Rh, Rb, Rt, M, P [936-954] CA ORF 106 2533AAGGCUGUUGCCAUCUCCA 2681 UGGAGAUGGCAACAGCC D, Rt [1127-1145] UU ORF 1072534 AUGACUUCGUGCGCAGCAA 2682 UUGCUGCGCACGAAGUC Rh, Rt, M [660-678] AUORF 108 2535 UCAGGCAAGAAGGACCUGA 2683 UCAGGUCCUUCUUGCCU Rh, D[1250-1268] GA ORF 109 2536 CUCAUCAUCCUCAUGCCCA 2684 UGGGCAUGAGGAUGAUGRh, Rt, M [1028-1046] AG ORF 110 2537 CGCGACGAGGAGGUGCACA 2685UGUGCACCUCCUCGUCG Rh, D [536-554] CG ORF 111 2538 ACAACCGUGGCUUCAUGGA2686 UCCAUGAAGCCACGGUU Rh, Rt, M [888-906] GU ORF 112 2539UUGACCAGGACAUCUACGA 2687 UCGUAGAUGUCCUGGUC Rt [1326-1344] AA ORF 1132540 CAAGCUGCCCGAGGUCACA 2688 UGUGACCUCGGGCAGCU Rh, D [778-796] UG ORF114 2541 UCCCUGCUAUUCAUUGGGA 2689 UCCCAAUGAAUAGCAGG D [1424-1442] GA ORF115 2542 UAUUCAUUGGGCGCCUGGA 2690 UCCAGGCGCCCAAUGAA D [1431-1449] UA ORF116 2543 CUGCGCGACGAGGAGGUGA 2691 UCACCUCCUCGUCGCGC Rh, D [533-551] AGORF 117 2544 CUACGGGCGCGAGGAGCUA 2692 UAGCUCCUCGCGCCCGU D, M [1339-1357]AG ORF 118 2545 CGCGAGGAGCUGCGCAGCA 2693 UGCUGCGCAGCUCCUCG D, M[1346-1364] CG ORF 119 2546 ACACCUGGCUGGGCUGGGA 2694 UCCCAGCCCAGCCAGGU D[1186-1204] GU ORF 120 2547 UCUACGGGCGCGAGGAGCA 2695 UGCUCCUCGCGCCCGUAD, M [1338-1356] GA ORF 121 2548 UUCUUCAAGCCACACUGGA 2696UCCAGUGUGGCUUGAAG Rh, Rb, D [842-860] AA ORF 122 2549CCUGGGCGAGCUGCUGCGA 2697 UCGCAGCAGCUCGCCCA Rh, D [559-577] GG ORF 1232550 AAGAAGGCUGUUGCCAUCA 2698 UGAUGGCAACAGCCUUC Rt [1124-1142] UU ORF124 2551 CGACGGCAAGCUGCCCGAA 2699 UUCGGGCAGCUUGCCGU D [772-790] CG ORF125 2552 GACGGCAAGCUGCCCGAGA 2700 UCUCGGGCAGCUUGCCG Rh, D [773-791] UCORF 126 2553 UUCAUUGGGCGCCUGGUCA 2701 UGACCAGGCGCCCAAUG Rh, D[1433-1451] AA ORF 127 2554 AAGCGCAGCGCGCUGCAGA 2702 UCUGCAGCGCGCUGCGCRh, Rt [725-743] UU ORF 128 2555 CCUGGCCCACAAGCUCUCA 2703UGAGAGCUUGUGGGCCA Rh, D, P [1006-1024] GG ORF 129 2556ACGGCAAGCUGCCCGAGGA 2704 UCCUCGGGCAGCUUGCC Rh, D [774-792] GU ORF 1302557 UUUGACCAGGACAUCUACA 2705 UGUAGAUGUCCUGGUCA Rt [1325-1343] AA ORF131 2558 UGACUUCGUGCGCAGCAGA 2706 UCUGCUGCGCACGAAGU Rh, Rt, M [661-679]CA ORF 132 2559 AAGGACGUGGAGCGCACGA 2707 UCGUGCGCUCCACGUCC Rh, D[797-815] UU ORF 133 2560 UCCAUCAACGAGUGGGCCA 2708 UGGCCCACUCGUUGAUGRt, M [743-761] GA ORF 134 2561 CACCGACGGCAAGCUGCCA 2709UGGCAGCUUGCCGUCGG D, Rt [769-787] UG ORF 135 2562 ACGGGCGCGAGGAGCUGCA2710 UGCAGCUCCUCGCGCCC D, M [1341-1359] GU ORF 136 2563UCCGCGACAAGCGCAGCGA 2711 UCGCUGCGCUUGUCGCG D [717-735] GA ORF 137 2564UUGGGCGCCUGGUCCGGCA 2712 UGCCGGACCAGGCGCCC Rh, D [1437-1455] AA ORF 1382565 AUGGUGGACAACCGUGGCA 2713 UGCCACGGUUGUCCACC Rh, M [881-899] AU ORF139 2566 AUUGGGCGCCUGGUCCGGA 2714 UCCGGACCAGGCGCCCA Rh, D [1436-1454] AUORF 140 2567 UACGGGCGCGAGGAGCUGA 2715 UCAGCUCCUCGCGCCCG D, M [1340-1358]UA ORF 141 2568 AUGCACCGGACAGGCCUCA 2716 UGAGGCCUGUCCGGUGC Rh, Rb, Rt, P[938-956] AU ORF 142 2569 UUCCACCACAAGAUGGUGA 2717 UCACCAUCUUGUGGUGGRh, Rb, D, P [869-887] AA ORF 143 2570 UUCCGCGACAAGCGCAGCA 2718UGCUGCGCUUGUCGCGG D [716-734] AA ORF 144 2571 UACCAGGCCAUGGCCAAGA 2719UCUUGGCCAUGGCCUGG Rh, D [392-410] UA ORF 145 2572 AAACACCUGGCUGGGCUGA2720 UCAGCCCAGCCAGGUGU D [1184-1202] UU ORF 146 2573 ACCGACGGCAAGCUGCCCA2721 UGGGCAGCUUGCCGUCG D [770-788] GU ORF 147 2574 AACACCUGGCUGGGCUGGA2722 UCCAGCCCAGCCAGGUG D [1185-1203] UU ORF 148 2575 UUCGUGCGCAGCAGCAAGA2723 UCUUGCUGCUGCGCACG Rh, D, M [665-683] AA ORF

The most active sequences were selected from further assays. From Table4 siRNA compounds SERPINH1_2, SERPINH1_6, SERPINH1_13, SERPINH1_45SERPINH1_45a, SERPINH1_51, SERPINH1_51a, SERPINH1_52 and SERPINH1_86were selected as preferred compounds (Tables 6-A and B).

TABLE 6-A Select siRNAs SEQ ID SEQ ID Activity Activity Activity siRNASEN AS 0.1 nM 0.5 nM 5 nM IC50 (nM) Length SERPINH1_2 60 127 65 48 7.008 19 SERPINH1_6 63 130 164 39 5 .019 19 SERPINH1_11 68 135 119 54 6.05 19 SERPINH1_13 69 136 91 24 4 19 SERPINH1_45 97 164 156 38 8 .07 19SERPINH1_45a 98 165 19 SERPINH1_51 101 168 68 39 5 .05 19 SERPINH1_52102 169 149 37 9 0.06 19 SERPINH1_86 123 190 121 61 0.27 19

TABLE 6B SEQ ID Activity Activity Activity Activity Activity ActivityActivity siRNA SEN SEQ ID AS 0.026 nM 0.077 nM 0.23 nM 0.69 nM 2.1 nM6.25 nM 25 nM SERPINH1_45 97 164 102 81 55 41 28 22 16 SERPINH1_45a 98165 107 98 84 69 36 24 16

From Table 5 siRNA compounds SERPINH1_4, SERPINH1_12, SERPINH1_18,SERPINH1_30, SERPINH1_58 and SERPINH1_88 were selected as preferredcompounds (Table 7).

TABLE 7 Select siRNAs SEQ ID SEQ ID Activity Activity Activity siRNA NOSEN NO AS 0.1 nM 0.5 nM 5 nM IC50 (nM) Length SERPINH1_4 195 220 60 35 5.006 19 SERPINH1_12 196 221 54 42 8 .065 19 SERPINH1_18 197 222 139 43 919 SERPINH1_30 199 224 146 49 9 0.093 19 SERPINH1_58 208 233 na na 8 19SERPINH1_88 217 242 105 43 9 19

Example 21: Animal Model Systems of Fibrotic Conditions

Testing the active siRNAs of the description may be done in predictiveanimal models. Rat diabetic and aging models of kidney fibrosis includeZucker diabetic fatty (ZDF) rats, aged fa/fa (obese Zucker) rats, agedSprague-Dawley (SD) rats, and Goto Kakizaki (GK) rats; GK rats are aninbred strain derived from Wistar rats, selected for spontaneousdevelopment of NIDDM (diabetes type II). Induced models of kidneyfibrosis include the permanent unilateral ureteral obstruction (UUO)model which is a model of acute interstitial fibrosis occurring inhealthy non-diabetic animals; renal fibrosis develops within daysfollowing the obstruction. Another induced model of kidney fibrosis is5/6 nephrectomy.

Two models of liver fibrosis in rats are the Bile Duct Ligation (BDL)with sham operation as controls, and CC14 poisoning, with olive oil fedanimals as controls, as described in the following references:Lotersztajn S, et al Hepatic Fibrosis: Molecular Mechanisms and DrugTargets. Annu Rev Pharmacol Toxicol. 2004 Oct. 7; Uchio K, et al.,Down-regulation of connective tissue growth factor and type I collagenmRNA expression by connective tissue growth factor antisenseoligonucleotide during experimental liver fibrosis. Wound Repair Regen.2004 January-February; 12(1):60-6; Xu X Q, et al., Molecularclassification of liver cirrhosis in a rat model by proteomics andbioinformatics Proteomics. 2004 October; 4(10):3235-45.

Models for ocular scarring are well known in the art e.g. Sherwood M Bet al., J Glaucoma. 2004 October; 13(5):407-12. A new model of glaucomafiltering surgery in the rat; Miller M H et al., Ophthalmic Surg. 1989May; 20(5):350-7. Wound healing in an animal model of glaucomafistulizing surgery in the Rb; vanBockxmeer F M et al., Retina. 1985Fall-Winter; 5(4): 239-52. Models for assessing scar tissue inhibitors;Wiedemann P et al., J Pharmacol Methods. 1984 August; 12(1): 69-78.Proliferative vitreoretinopathy: the Rb cell injection model forscreening of antiproliferative drugs.

Models of cataract are described in the following publications: Zhou etal., 2002. Invest Ophthalmol Vis Sci.43:2293-300; Wang et al. 2004 CurrEye Res. 29:51-58.

The compounds of Table 5 and Table 4 are tested in these models offibrotic conditions, in which it is found that they are effective intreating liver fibrosis and other fibrotic conditions.

Model Systems of Glaucoma

Testing the active siRNA of the description for treating or preventingglaucoma is performed in a rat animal model for optic nerve crushdescribed, for example, in: Maeda et al., 2004, InvestigativeOphthalmology and visual Science (IOVS), 45:851. Specifically, for opticnerve transection the orbital optic nerve (ON) of anesthetized rats isexposed through a supraorbital approach, the meninges severed and allaxons in the ON transected by crushing with forceps for 10 seconds, 2 mmfrom the lamina cribrosa.

Nucleic acid molecules as disclosed herein are tested in this animalmodel and the results show that these siRNA compounds are useful intreating and/or preventing glaucoma.

Rat Optic Nerve Crush (ONC) Model: Intravitreal siRNA Delivery and EyeDrop Delivery

For optic nerve transsection the orbital optic nerve (ON) ofanesthetized rats is exposed through a supraorbital approach, themeninges severed and all axons in the ON transected by crushing withforceps for 10 seconds, 2 mm from the lamina cribrosa.

The siRNA compounds are delivered alone or in combination in 5 uL volume(10 ug/uL) as eye drops. Immediately after optic nerve crush (ONC), 20ug/10 ul test siRNA or 10 ul PBS is administered to one or both eyes ofadult Wistar rats and the levels of siRNA taken up into the dissectedand snap frozen whole retinae at 5 h and 1 d, and later at 2 d, 4 d, 7d, 14 d and 21 d post injection is determined. Similar experiments areperformed in order to test activity and efficacy of siRNA administeredvia eye drops.

Model Systems of Ischemia Reperfusion Injury Following LungTransplantation in Rats

Lung ischemia/reperfusion injury is achieved in a rat animal model asdescribed in Mizobuchi et al., 2004 J Heart Lung Transplantation, 23 andin Kazuhiro et al., 2001 Am. J. Respir. Cell Mol Biol, 25:26-34.

Specifically, after inducing anesthesia with isofluorane, the trachea iscannulated with a 14-gauge Teflon catheter and the rat is mechanicallyventilated with rodent ventilator using 100% oxygen, at a rate of 70breaths per minute and 2 cm H₂O of positive end-respiratory pressure.The left pulmonary artery, veins and main stem bronchus are occludedwith a Castaneda clamp. During the operation, the lung is kept moistwith saline and the incision is covered to minimize evaporative losses.The period of ischemia is 60 minutes long. At the end of the ischemicperiod the clamp is removed and the lung is allowed to ventilate andreperfuse for further 4 h, 24 h, and 5 d post induction of lungischemia. At the end of the experiment, the lungs are gently harvestedand either frozen for RNA extraction or fixed in glutaraldehyde cocktailfor subsequent histological analysis.

The Bleomycin Animal Model as a Model for Idiopathic Pulmonary Fibrosis(IPF)

Testing feasibility of lung and liver delivery of vitamin A-Coatsomeformulated siRNA administered by intravenous injection and intratrachealadministration of siRNA-vitaminA-Coatsome complex to a healthy mice andbleomycine-treated mice

Objective: To test two administration routes for feasibility of vitaminA-Coatsome formulated siRNA delivery to normal and fibrotic mouse lungs.The main hypothesis to be tested in the current study is whethersystemic administration of vitamin A-Coatsome formulated modified siRNAprovides efficient uptake and cell-specific distribution in the fibroticand normal mouse lungs. Intratracheal route of vitaminA-Coatsomeformulated modified siRNA will be tested in parallel. siRNA detectionand cell-specific distribution in the lungs and liver will be performedby in situ hybridization (ISH)

The Bleomycin model of pulmonary fibrosis has been well developed andcharacterized over the last three decades (Moeller, et al., 2008, Int JBiochem Cell Biol, 40:362-82; Chua et al., 2005, Am J Respir Cell MolBiol, 33:9-13). Histological hallmarks, such as intra-alveolar buds,mural incorporation of collagen and obliteration of alveolar space arepresent in BLM-treated animals similar to IPF patients. Early studiesdemonstrated that C57/Bl mice were consistently prone to BLM-inducedlung fibrosis, whereas Balb/C mice were inherently resistant. Dependingon the route of administration, different fibrotic pattern develops.Intratracheal instillation of BLM results in bronchiocentric accentuatedfibrosis, whereas intravenous or intraperitoneal administration inducessubpleural scarring similar to human disease (Chua et al. ibid.). Amouse model of usual interstitial pneumonia (UIP) is used. This modelshows a heterogenous distribution of fibroproliferation, distributedmainly subpleurally, forming similar lesions to those observed in thelungs of patients with idiopathic pulmonary fibrosis (IPF) (Onuma, etal., 2001 J Exp Med 194:147-56, and Yamaguchi et al., 1988 Nature336:244-46). UIP will be induced by intraperitoneal injection ofbleomycin every other day for 7 days for a constant composition ofsubpleural fibroproliferation in the mouse lung (Swiderski et al. 1998Am J Pathol 152: 821-28, and Shimizukawa et al., 2003 Am J Physiol LungCell Mol Physiol 284: L526-32,).

Vitamin A-loaded liposomes containing siRNA interact withretinol-binding protein (RBP) and provide efficient delivery to the HSCvia RBP receptor. These liposomes are efficiently taken up by an RBPreceptor-expressing activated myofibroblasts in the lungs ofbleomycin-treated mice.

Study Design

Mice—C57 Bl male

Starting N (BLM I.P.)—40 (6 for the first pilot group, 34 for the study,taking in consideration anticipated 25% mortality)

Starting N (Total)—60

Test siRNA: SERPINHI compounds disclosed herein.

TABLE 8 Groups: BLM dose, mg/kg siRNA BW, in BLM dose, Termination N(before 0.1 ml adm. mg/kg siRNA siRNA post last siRNA No saline routeBLM regime BW adm route regime siRNA adm administration) 1 0.75 I.P. dd0, 2, 4, 6 4.5 I.V. 2 daily  2 h 4 2 0.75 I.P. dd 0, 2, 4, 6 4.5 I.V. 2daily 24 h 4 3 0.75 I.P. dd 0, 2, 4, 6 2.25 I.T. 2 daily  2 h 4 4 0.75I.P. dd 0, 2, 4, 6 2.25 I.T. 2 daily 24 h 4 5 intact n/a 4.5 I.V. 2daily  2 h 4 6 intact n/a 4.5 I.V 2 daily 24 h 4 7 intact n/a 2.25 I.T 2daily  2 h 4 8 intact n/a 2.25 I.T. 2 daily 24 h 4 9 0.75 I.P. dd 0, 2,4, 6 n/a I.V. 2 daily  2 h 3 vehicle 10 0.75 I.P. dd 0, 2, 4, 6 n/aI/T/vehicle 2 daily 24 h 3 11 Intact n/a n/a intact n/a Any time 3

Bleomycin-Induced Pulmonary Fibrosis.

Pulmonary fibrosis of 12-wk-old female C57BL/6 mice will be induced byintraperitoneal instillation of bleomycin chlorate: 0.75 mg/kg bodyweight dissolved in 0.1 ml of saline every other day for 7 days, on days0, 2, 4, and 6.

Pilot Evaluation of the Establishment of Fibrosis.

The mice (N=30) are subjected to BLM treatment in groups, to allow for aone week time interval between the first treated group (N=5) and therest of the animals. On day 14, two mice from the first group aresacrificed and the lungs harvested for the fast HE stain and quickhistopathological evaluation of fibrosis. When lung fibrosis isconfirmed, the remaining rats are sorted into the groups and treatedwith siRNA on Day 14 after the first BLM treatment. In case that nosufficient fibrosis develops in the lungs by day 14, the remaining micefrom the first treated group are sacrificed on day 21, followed by quickhistopathology evaluation of fibrosis. The rest of the animals aretreated with test siRNA complex starting from day 21 after the BLMtreatment.

siRNA Administration.

On day 14 or day 21 after the first BLM administration (TBD during thestudy, based on pilot evaluation of establishment of fibrosis), theanimals are group sorted, according to BW. The animals from groups 1 and2 are administered intravenously (tail vein injection) withsiRNA/vitA/Coatsome complex, at an siRNA concentration of 4.5 mg/kg BW.Intact animals of the same age (Groups 5 and 6) are treated in the samemanner. BLM treated animals (Group 9) will be used as vitA-coatsomevehicle control). In 24 hours, the injection is repeated to all theanimals, as above.

The BLM animals from the groups 3 and 4, and intact mice from groups 7and 8 are anesthetized with isoflurane and subjected to intratrachealinstillation of 2.25 mg/kg BW siRNA formulated in vitA-loaded liposomes.Mice from the BLM group 10 are administered with vitA/Coatsome vehicleonly. The intratracheal instillation is repeated after 24 hours.

Study Termination.

The animals from the groups 1, 3, 5, 7, 9 are sacrificed at 2 hoursafter the second siRNA complex injection or instillation. The animalsfrom the groups 2, 4, 6, 8, 10 are sacrificed at 24 hours after thesecond siRNA complex injection or instillation.

Upon animals sacrifice, the mice are perfused transcardially with 10%neutral buffered formalin. The lungs are inflated with 0.8-1.0 ml of 10%NBF, and the trachea ligated. The lungs are excised and fixed for 24 hin 10% NBF. The liver is harvested from each animal and fixed in 10% NBFfor 24 h.

Sectioning and evaluation.

Consequent sections are prepared from the lungs and livers. Firstconsequent section are stained with hematoxylin and eosin for assessmentof lung and liver morphology, second section are stained with Sirius Red(trichrome) to identify collagen The third consequent sections aresubjected to in situ hybridization (ISH) for detection of siRNA.

Example 22: In Vivo Anti-Pulmonary-Fibrosis Activity of siRNA-ContainingVA-Bound Liposome

(1) Induction of Pulmonary Fibrosis and Administration of Drug

Male S-D rats (8 rats/group, 8 weeks old, Charles River LaboratoriesJapan, Inc.) were administered once with 0.45 mg bleomycin (BLM)dissolved in 0.1 mL of saline into the lung by intratracheal cannulation(MicroSprayer, Penn-Century, Inc.) under anesthesia, to produce ableomycin pulmonary fibrosis model. With this method, a significantfibrosis occurs in the lung generally after approximately 2 weeks. Theliposome formulation (1.5 mg/kg as an amount of siRNA, 1 ml/kg involume, i.e., 200 μl for a rat of 200 g) or PBS (1 ml/kg in volume) wasadministered to the rats via the tail vein, starting from the 2 weeksafter the bleomycin administration, for total of ten times (every otherday). The rats were sacrificed at two days post last treatment,histological investigation of the lung tissue was performed (see FIG.30). One way ANOVA and Bonferroni multi comparison test was used for theevaluation of statistically-significant difference.

The composition of the liposome wasHEDC/S-104/DOPE/Cholesterol/PEG-DMPE/diVA-PEG-diVA (20:20:30:25:5:2Molar %. Details of siRNA were as follows:

S strand: 5′-idAB-rG-rA-rG-rA-rC-rA-rC-rA-rU-rG-rG-rG-rU-rG-25rC-25rU-25rA-25rU-25rA-C3-P-3′ GS strand:5′-mU-rA-mU-rA-mG-rC-25rA-rC-mC-rC-mA-rU-mG-rU- mG-rU-mC-rU-mC-C3-C3-3′wherein: rX represents ribonucleotides; mX represents 2′-O-methylribonucleotides; 25rX represents ribonucleotides with 2′-5′ linkages; C3represents a 1,3-propanediol spacer; idAB represents inverted1,2-dideoxy-D-ribose; P represents a phosphate group on the 3′-terminus.The 3′-terminus C3 is introduced by support-loaded 1,3-propanediolspacer. The 3′-terminus phosphate group (P) is introduced by the use ofsupport-loaded diethyl sulfonyl (Pi) spacer.

(2) Histological Investigation

A part of the removed lung was formalin-fixed in accordance with aroutine method, and subjected to azan staining (azocarmine, aniline blueorange G solution). As shown by the results of the azan staining in FIG.31, in the PBS administration group (Disease), a noticeable fibroticimage characterized by enlargement of interstice due to a large quantityof blue-stained collagenous fibrils was observed, whereas in theformulation administration group (Treatment), fibrosis were apparentlysuppressed.

As shown by the results of histological scoring (T. Ashcroft score) inFIG. 32, in the formulation administration group (Treatment), fibrosisscore was significantly decreased.

Example 23: In-Vivo Reduction of HSP47 mRNA (DMN Model)

In vivo activity of the HSP47 liposomes of Example 22 was evaluated inthe short-term liver damage model (referred to as the Quick Model). Inthis model, the short-term liver damage induced by treatment with ahepatotoxic agent such as dimethylnitrosamine (DMN) is accompanied bythe elevation of HSP47 mRNA levels. To induce these changes, maleSprague-Dawley rats were injected intraperitoneally with DMN on sixconsecutive days. At the end of the DMN treatment period, the animalswere randomized to groups based upon individual animal body weight. Theliposome sample was administered as a single IV dose (0.375 or 0.75mg/kg, reflecting the siRNA dose) one hour after the last injection ofDMN. One day later, liver lobes were excised and both HSP47 and GAPDHmRNA levels were determined by quantitative RT-PCR (TaqMan) assay. HSP47mRNA levels were normalized to GAPDH levels. As shown in FIG. 33, robustand dose-dependent mRNA reduction for HSP47 was detected in liver. Aftera single dose of 0.75 mg/kg of ND-L02-0101, 80% reduction of HSP47 mRNAwas observed. Even at the lower dose of 0.375 mg/kg, significantknockdown of 40% was observed.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference.

Applicants reserve the right to physically incorporate into thisapplication any and all materials and information from any sucharticles, patents, patent applications, or other physical and electronicdocuments.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the description disclosedherein without departing from the scope and spirit of the description.Thus, such additional embodiments are within the scope of the presentdescription and the following claims. The present description teachesone skilled in the art to test various combinations and/or substitutionsof chemical modifications described herein toward generating nucleicacid constructs with improved activity for mediating RNAi activity. Suchimproved activity can include improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying nucleic acidmolecules with improved RNAi activity.

The descriptions illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “a” and “an” and “the” and similar referents in the context ofdescribing the description (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.The terms “comprising”, “having,” “including,” containing”, etc. shallbe read expansively and without limitation (e.g., meaning “including,but not limited to,”). Recitation of ranges of values herein are merelyintended to serve as a shorthand method of referring individually toeach separate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the description and does not pose alimitation on the scope of the description unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the description.Additionally, the terms and expressions employed herein have been usedas terms of description and not of limitation, and there is no intentionin the use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe description claimed. Thus, it should be understood that although thepresent description has been specifically disclosed by preferredembodiments and optional features, modification and variation of thedescriptions embodied therein herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this description.

The description has been described broadly and generically herein. Eachof the narrower species and subgeneric groupings falling within thegeneric disclosure also form part of the description. This includes thegeneric description of the description with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.Other embodiments are within the following claims. In addition, wherefeatures or aspects of the description are described in terms of Markushgroups, those skilled in the art will recognize that the description isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

What is claimed:
 1. A method for treating fibrosis in a subject in needthereof, the method comprising administering an effective amount of apharmaceutical composition to the subject, wherein the pharmaceuticalcomposition comprising a drug carrier and a double-stranded nucleic acidmolecule, wherein the drug carrier comprises a cationic lipid and aretinoid molecule, and wherein the double-stranded nucleic acid moleculecomprises the modified nucleic acid molecules having a structure (A2)set forth below: (A2) 5′ N¹-(N)_(x)-Z 3′ (antisense strand) 3′Z′-N²-(N′)_(y)-z″ 5′ (sense strand)

wherein each of N¹, N², N and N′ is an unmodified or modifiedribonucleotide, or an unconventional moiety; wherein each of (N)_(x) and(N′)_(y) is an oligonucleotide in which each consecutive N or N′ isjoined to the adjacent N or N′ by a covalent bond; wherein each of x andy is independently an integer between 17 and 39; wherein the sequence of(N′)_(y) has complementarity to the sequence of (N)_(x) and (N)_(x)comprises an antisense sequence to the mRNA coding sequence for humanhsp47 exemplified by SEQ ID NO:1; wherein N¹ is covalently bound to(N)_(x) and is mismatched to the target RNA or is a complementary DNAmoiety to the target RNA; wherein each of Z and Z′ is independentlypresent or absent, but if present is independently 1-5 consecutivenucleotides, consecutive non-nucleotide moieties or a combinationthereof covalently attached at the 3′-terminus of the strand in which itis present; wherein z″ may be present or absent, but if present is acapping moiety covalently attached at the 5′-terminus of N²—(N′)_(y);and wherein N¹ is a moiety selected from the group consisting of naturalor modified uridine, deoxyribouridine, ribothymidine,deoxyribothymidine, adenosine or deoxyadenosine.
 2. The method of claim1, wherein the cationic lipid is a molecule with the structure


3. The method of claim 1, wherein the cationic lipid is a molecule withthe structure


4. The method according to claim 1, wherein the pharmaceuticalformulation is parenterally administered.
 5. The method of claim 1,wherein the fibrosis is liver fibrosis.
 6. The method of claim 5,wherein the liver fibrosis is selected from the group consisting ofnon-alcoholic steatohepatitis (NASH); hepatitis; hepatic fibrosis;chronic hepatitis C virus (HCV) infection; hepatic cirrhosis; chronichepatic damage; and liver cancer.
 7. The method of claim 1, wherein thefibrosis is pulmonary fibrosis.
 8. The method of claim 7, wherein thefibrosis is selected from the group consisting of pulmonary fibrosisincluding idiopathic pulmonary fibrosis, interstitial lung disease,radiation pneumonitis leading to pulmonary fibrosis; and fibrotic lungdisease.
 9. The method of claim 1, wherein the fibrosis is kidneyfibrosis.
 10. The method of claim 9, wherein the kidney fibrosis ischronic renal failure or diabetic nephropathy.
 11. The method of claim1, wherein the fibrosis is peritoneal fibrosis.
 12. The method of claim11, wherein the peritoneal fibrosis is peritoneal sclerosis.
 13. Themethod of claim 1, wherein the fibrosis is pancreatic fibrosis.
 14. Themethod of claim 13, wherein the pancreatic fibrosis is pancreatitis,pancreatic fibrosis, or pancreatic cancer.
 15. The method of claim 1,wherein the fibrosis is cardiac fibrosis.
 16. The method of claim 15,wherein the cardiac fibrosis is myocardial fibrosis.
 17. The method ofclaim 1, wherein the fibrosis is intestinal fibrosis.
 18. The method ofclaim 1, wherein the fibrosis is fibrosis of the eye.
 19. The method ofclaim 18, wherein the fibrosis of the eye is selected from the groupconsisting of ocular scarring including proliferative vitreoretinopathy(PVR) and scarring resulting from surgery to treat cataract or maculardegeneration, glaucoma, Grave's ophthalmopathy; ocular cicatricialpemphigoid, and drug induced ergotism.
 20. The method of claim 1,wherein the fibrosis is skin fibrosis.
 21. The method of claim 20,wherein the skin fibrosis including scleroderma, keloids andhypertrophic scars scleroderma; psoriasis; and Kaposi's sarcoma.
 22. Themethod of claim 1, wherein the fibrosis is fibrosis of bone.
 23. Themethod of claim 1, wherein the fibrosis is selected from the groupconsisting of myelofibrosis; myleoid leukemia; acute myelogenousleukemia; myelodysplastic syndrome; and myeloproferative syndrome. 24.The method of claim 1, wherein the fibrosis is inflammatory boweldisease of variable etiology.
 25. The method of claim 1, wherein thefibrosis is glioblastoma in Li-Fraumeni syndrome or sporadicglioblastoma.
 26. The method of claim 1, wherein the fibrosis isgynecological cancer.
 27. The method of claim 1, wherein the fibrosis isHansen's disease.
 28. The method of claim 1, wherein the fibrosis iscollagenous colitis.
 29. The method of claim 1, wherein the fibrosis isfibrillogenesis.
 30. The method of claim 1, wherein the fibrosis isfibrosis of vocal cords.
 31. The method of claim 30, wherein thefibrosis is selected from the group consisting of vocal cord scarring,vocal cord mucosal fibrosis, and laryngeal fibrosis.